Methods of using ox40 ligand encoding polynucleotides

ABSTRACT

The disclosure relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotide molecules comprising an mRNA encoding an OX40L polypeptide. Also provided is a method for activating T cells or increasing the number of NK cells in a subject in need thereof.

RELATED APPLICATIONS

This application is a Continuation of Application PCT/US2016/068552filed on Dec. 23, 2016. Application PCT/US2016/068552 claims the benefitof U.S. Provisional Application 62/387,168 filed on Dec. 23, 2015 andU.S. Provisional Application 62/290,413 filed on Feb. 2, 2016. Theentire contents of the above-referenced patent applications areincorporated herein by this reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 1, 2018, isnamed “SeqListing_MDN705PCCN” and is 59,483 bytes in size. The SequenceListing is being submitted by EFS Web and is hereby incorporated byreference into the specification.

BACKGROUND

Cancer is a disease characterized by uncontrolled cell division andgrowth within the body. In the United States, roughly a third of allwomen and half of all men will experience cancer in their lifetime.Polypeptides are involved in every aspect of the disease includingcancer cell biology (carcinogenesis, cell cycle suppression, DNA repairand angiogenesis), treatment (immunotherapy, hormone manipulation,enzymatic inhibition), and/or diagnosis and determination of cancer type(molecular markers for breast, prostate, colon and cervical cancer forexample). With the host of undesired consequences brought about bystandard treatments such as chemotherapy and radiotherapy used today,genetic therapy for the manipulation of disease-related peptides andtheir functions provides a more targeted approach to disease diagnosis,treatment and management. However, gene therapy poses multiplechallenges including undesirable immune response and safety concern dueto the incorporation of the gene at random locations within the genome.

Various methods of treating cancer are under development. For example,dendritic cell (DC) vaccines have been studied as a possible anti-cancertherapy. However, DC vaccines require multiple steps of isolating DCsfrom a subject, ex vivo manipulation of DCs to prime the cells for tumorantigen presentation, and subsequent administration of the manipulatedDCs back into the subject. Further, it is reported that the overallclinical response rates for DC vaccines remain low and the ability of DCvaccines to induce cancer regression remains low. See, e.g., Kalkinskiet al., “Dendritic cell-based therapeutic cancer vaccines: what we haveand what we need,” Future Oncol. 5(3):379-390 (2009).

BRIEF SUMMARY

The present disclosure relates to compositions and methods foractivating an immune response in a subject. One aspect of the disclosureprovides a method of activating T cells in a subject in need thereofcomprising administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Inanother aspect, the activated T cells reduce or decrease the size of atumor or inhibit growth of a tumor in the subject.

Another aspect of the disclosure provides a method of increasing thenumber of Natural Killer (NK) cells in a subject in need thereofcomprising administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Inanother aspect, the increased NK cells reduce or decrease the size of atumor or inhibit growth of a tumor in the subject.

In some embodiments, the disclosure provides a method for activating Tcells and increasing the number of NK cells in a subject in need thereofcomprising administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide.

In another embodiment, administering to the subject an effective amountof a polynucleotide comprising an mRNA encoding an OX40L polypeptidefurther induces IL-2 release. In another embodiment, administering tothe subject an effective amount of a polynucleotide comprising an mRNAencoding an OX40L polypeptide further induces IL-4 release. In anotherembodiment, administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide furtherinduces IL-21 release.

In some embodiments, administering to the subject an effective amount ofa polynucleotide comprising an mRNA encoding an OX40L polypeptideinduces T cell proliferation. In other embodiments, administering to thesubject an effective amount of a polynucleotide comprising an mRNAencoding an OX40L polypeptide induces T cell infiltration in the tumoror increases the number of tumor infiltrating T cells. In certainembodiments, administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide inducesa memory T cell response. In some embodiments, the activated T cellscomprise CD4⁺ T cells. In other embodiments the activated T cellscomprise CD8⁺ T cells. In other embodiments, the activated T cellscomprise both CD4⁺ T cell and CD8 T cells. In some embodiments, thenumber of NK cells is increased at least about two-fold, at least aboutthree-fold, at least about four-fold, at least about five-fold, at leastabout six-fold, at least about seven-fold, at least about eight-fold, atleast about nine-fold, or at least about ten-fold.

In some embodiments of the disclosure, the polynucleotide comprises atleast one chemically modified nucleoside as described herein. In oneembodiment, the at least one chemically modified nucleoside comprisestwo or more combinations thereof. In another embodiment, the at leastone chemically modified nucleoside is selected from the group consistingof pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine (s2U),4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methoxyuridine, 2′-O-methyl uridine,1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U),5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4′-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A),2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and2,6-Diaminopurine, (I), 1-methylinosine (m1I), wyosine (imG),methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine(m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, and two or more combinations thereof.

In another embodiment of the disclosure, the nucleosides in the mRNA arechemically modified by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or 100%. In another aspect, thechemically modified nucleosides in the mRNA are selected from the groupconsisting of uridine, adenine, cytosine, guanine, and any combinationthereof.

In one embodiment, the uridine nucleosides in the mRNA are chemicallymodified by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100%. In another embodiment, the adeninenucleosides in the mRNA are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%. In another embodiment, the cytosine nucleosides in the mRNA arechemically modified by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or 100%. In another embodiment,the guanine nucleosides in mRNA are chemically modified by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

The present disclosure further provides a method of activating T cellsin a subject in need thereof or increasing the number of NK cells in asubject in need thereof comprising administering to the subject aneffective amount of a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the mRNA encoding the OX40L polypeptide is anopen reading frame. In one aspect, the OX40L polypeptide comprises anamino acid sequence at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to a sequence listed in Table 1, wherein the amino acidsequence is capable of binding to an OX40 receptor.

Another aspect of the disclosure provides a method of activating T cellsin a subject in need thereof or increasing the number of NK cells in asubject in need thereof comprising administering to the subject aneffective amount of a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the polynucleotide further comprises anucleic acid sequence comprising a miRNA binding site. In oneembodiment, the miRNA binding site binds to miR-122. In a particularembodiment, the miRNA binding site binds to miR-122-3p or miR-122-5p.

In one aspect of the disclosure, the miRNA binding site comprises anucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, or 100% identical to SEQ ID NO: 26, wherein the miRNA binding sitebinds to miR-122. In another aspect, the miRNA binding site comprises anucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, or 100% identical to SEQ ID NO: 24, wherein the miRNA binding sitebinds to miR-122. In another aspect, the miRNA binding site comprises anucleotide sequence that binds to SEQ ID NO: 22.

Another aspect of the disclosure provides a method of activating T cellsin a subject in need thereof or increasing the number of NK cells in asubject in need thereof comprising administering to the subject aneffective amount of a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the polynucleotide further comprises a 5′untranslated region (UTR). In some embodiments, the 5′ UTR comprises anucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence listed in Table 3.

In another aspect, the polynucleotide used in the methods of thedisclosure further comprises a 3′ UTR. In some embodiments, the 3′ UTRcomprises a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a sequence listed in Table 4.

In another aspect of the disclosure, the miRNA binding site is insertedwithin the 3′ UTR. In some aspects, the polynucleotide further comprisesa spacer sequence between the open reading frame and the miRNA bindingsite. In one aspect, the spacer sequence comprises at least about 10nucleotides, at least about 20 nucleotides, at least about 30nucleotides, at least about 40 nucleotides, at least about 50nucleotides, at least about 60 nucleotides, at least about 70nucleotides, at least about 80 nucleotides, at least about 90nucleotides, or at least about 100 nucleotides.

In some embodiments of the disclosure, the polynucleotide furthercomprises a 5′ terminal cap. In one embodiment, the 5′ terminal cap is aCap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

In another embodiment, the polynucleotide further comprises a 3′polyadenylation (polyA tail).

In some embodiments of the disclosure, the polynucleotide comprises atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or at least ten miRNAbinding sites.

In another embodiment, the polynucleotide is codon optimized. In oneaspect, the polynucleotide is codon optimized for expression in amammal. In a particular embodiment, the polynucleotide is codonoptimized for expression in a human.

In some embodiments, the polynucleotide is in vitro transcribed (IVT).In other embodiments, the polynucleotide is chimeric. In otherembodiments, the polynucleotide is circular.

Another aspect of the disclosure provides a method of activating T cellsin a subject in need thereof or increasing the number of NK cells in asubject in need thereof comprising administering to the subject aneffective amount of a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the OX40L polypeptide is fused to one or moreheterologous polypeptides. In one aspect, the one or more heterologouspolypeptides increase a pharmacokinetic property of the OX40Lpolypeptide

In another aspect of the disclosure, the polynucleotide furthercomprises a second open reading frame encoding a second polypeptide.

In some embodiments of the present disclosure, the methods furthercomprise administering a second polynucleotide. In one embodiment, thesecond polynucleotide comprises a second open reading frame encoding asecond polypeptide. In another embodiment, the second open reading frameis a second mRNA. In some embodiments, the second mRNA comprises atleast one chemically modified nucleoside.

In another embodiment of the disclosure, the polynucleotide isformulated with a delivery agent. In some embodiments, the deliveryagent comprises a lipidoid, a liposome, a lipoplex, a lipidnanoparticle, a polymeric compound, a peptide, a protein, a cell, ananoparticle mimic, a nanotube, or a conjugate. In one embodiment, thedelivery agent is a lipid nanoparticle. In another embodiment, the lipidnanoparticle comprises the lipid selected from the group consisting ofDLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA,DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, amino alcohol lipids, KL22,and combinations thereof.

In some embodiments of the disclosure, the polynucleotide is formulatedfor in vivo delivery. In one aspect, the polynucleotide is formulatedfor subcutaneous, intravenous, intraperitoneal, intratumoral,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial,intraventricular, oral, inhalation spray, topical, rectal, nasal,buccal, vaginal, intratumoral, or implanted reservoir intramuscular,subcutaneous, intratumoral, or intradermal delivery. In another aspect,the polynucleotide is administered subcutaneously, intravenously,intraperitoneally, intratumorally, intramuscularly, intra-articularly,intra-synovially, intrasternally, intrathecally, intrahepatically,intralesionally, intracranially, intraventricularly, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir.

In some embodiments, the methods of the present disclosure treat acancer. In one aspect, the cancer is selected from the group consistingof adrenal cortical cancer, advanced cancer, anal cancer, aplasticanemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis,brain tumors, brain cancer, breast cancer, childhood cancer, cancer ofunknown primary origin, Castleman disease, cervical cancer, colon/rectalcancer, endometrial cancer, esophagus cancer, Ewing family of tumors,eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors,gastrointestinal stromal tumors, gestational trophoblastic disease,Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal andhypopharyngeal cancer, acute lymphocytic leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia,chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma(HCC), non-small cell lung cancer, small cell lung cancer, lungcarcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiplemyeloma, myelodysplastic syndrome, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oralcavity and oropharyngeal cancer, osteosarcoma, ovarian cancer,pancreatic cancer, penile cancer, pituitary tumors, prostate cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma inadult soft tissue, basal and squamous cell skin cancer, melanoma, smallintestine cancer, stomach cancer, testicular cancer, throat cancer,thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvarcancer, Waldenstrom macroglobulinemia, Wilms tumor, secondary cancerscaused by cancer treatment, and any combination thereof.

In some aspects of the disclosure, the polynucleotide is delivered by adevice comprising a pump, patch, drug reservoir, short needle device,single needle device, multiple needle device, micro-needle device, jetinjection device, ballistic powder/particle delivery device, catheter,lumen, cryoprobe, cannula, microcanular, or devices utilizing heat, RFenergy, electric current, or any combination thereof.

In one aspect, the polynucleotide is administered intratumorally to thesubject at a unit dose of at least about 10 μg, at least about 12.5 μg,or at least about 15 μg. In another aspect, the effective amount isbetween about 0.10 μg/kg and about 1000 mg/kg.

In a particular aspect of the disclosure, the number of NK cells isincreased at least five-fold at 24 hours after the administration.

In another aspect, the tumor growth is inhibited at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, or about 100% after theadministration compared to a control polynucleotide that does not encodean OX40L polypeptide.

In one aspect of the disclosure, the methods of the disclosure furthercomprise administering a PD-1 antagonist to the subject. In someaspects, the PD-1 antagonist is an antibody or an antigen-bindingportion thereof that specifically binds to PD-1. In a particular aspect,the PD-1 antagonist is a monoclonal antibody. In some aspects, the PD-1antagonist is selected from the group consisting of Nivolumab,Pembrolizumab, Pidilizumab, and any combination thereof.

In another aspect, the methods of the disclosure further compriseadministering a PD-L1 antagonist to the subject. In some aspects, thePD-L1 antagonist is an antibody or an antigen-binding portion thereofthat specifically binds to PD-L1. In a particular aspect, the PD-L1antagonist is a monoclonal antibody. In some aspects, the PD-L1antagonist is selected from the group consisting of Durvalumab,Avelumab, MEDI473, BMS-936559, Atezolizumab, and any combinationthereof.

In another aspect, the methods of the disclosure further compriseadministering a CTLA-4 antagonist to the subject. In some aspects, theCTLA-4 antagonist is an antibody or an antigen-binding portion thereofthat specifically binds to CTLA-4. In a particular aspect, the CTLA-4antagonist is a monoclonal antibody. In some aspects, the CTLA-4antagonist is selected from the group consisting of Ipilimumab,Tremelimumab, and any combination thereof.

EMBODIMENTS

E1. A method of activating T cells in a subject in need thereofcomprising administering to the subject an effective amount of apolynucleotide comprising an mRNA encoding an OX40L polypeptide, whereinthe activated T cells reduce or decrease the size of a tumor or inhibitgrowth of a tumor.

E2. The method of embodiment E1 wherein the T cell activation comprisesinducing T cell proliferation.

E3. The method of embodiment E1 or E2, wherein the T cells activationcomprises inducing T cell infiltration in the tumor or increasing thenumber of tumor-infiltrating T cells.

E4. The method of any one of embodiments E1 to E3, wherein the T cellactivation comprises inducing a memory T cell response.

E5. The method of any one of embodiments E1 to E4, wherein the activatedT cells comprises CD4⁺ T cells, CD8⁺ T cells, or both.

E6. The method of any one of embodiments E1 to E5, wherein theadministering further increases the number of NK cells in the subject.

E7. A method of increasing the number of Natural Killer (NK) cells in asubject in need thereof comprising administering to the subject aneffective amount of a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the increased NK cells reduce or decrease thesize of a tumor or inhibit growth of a tumor.

E8. The method of any one of embodiments E1 to E7, wherein the T cellactivation comprises inducing IL-2 release, IL-4 release, IL-21,release, or any combination thereof.

E9. The method of any one of embodiments E6 to E8, wherein the number ofNK cells is increased at least about two-fold, at least aboutthree-fold, at least about four-fold, at least about five-fold, at leastabout six-fold, at least about seven-fold, at least about eight-fold, atleast about nine-fold, or at least about ten-fold.

E10. The method of any one of embodiments E1 to E9, wherein thepolynucleotide comprises at least one chemically modified nucleoside.

E11. The method of embodiment E10, wherein the at least one chemicallymodified nucleoside is selected from the group consisting ofpseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine (s2U),4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methoxyuridine, 2′-O-methyl uridine,1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U),5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4′-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A),2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and2,6-Diaminopurine, (I), 1-methylinosine (m1I), wyosine (imG),methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine(m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, and two or more combinations thereof.

E12. The method of any one of embodiments E1 to E11, wherein thenucleosides in the mRNA are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

E13. The method of any one of embodiments E10 to E12, wherein thechemically modified nucleosides in the mRNA are selected from the groupconsisting of uridine, adenine, cytosine, guanine, and any combinationthereof.

E14. The method of any one of embodiments E1 to E13, wherein the uridinenucleosides in the mRNA are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

E15. The method of any one of embodiments E1 to E14, wherein the adeninenucleosides in the mRNA are chemically modified by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%.

E16. The method of any one of embodiments E1 to E15, wherein thecytosine nucleosides in the mRNA are chemically modified by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or 100%.

E17. The method of any one of embodiments E1 to E16, wherein the guaninenucleosides in mRNA are chemically modified by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.

E18. The method of any one of embodiments E1 to E17, wherein the mRNAencoding the OX40L polypeptide is an open reading frame.

E19. The method of embodiment E18, wherein the OX40L polypeptidecomprises an amino acid sequence at least 50%, at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 2, wherein the amino acid sequenceis capable of binding to an OX40 receptor.

E20. The method of embodiment E18, wherein the OX40L polypeptidecomprises an amino acid sequence at least 50%, at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to SEQ ID NO: 1, wherein the amino acid sequenceis capable of binding to an OX40 receptor.

E21. The method of any one of embodiments E1 to E20, wherein thepolynucleotide further comprises a nucleic acid sequence comprising amiRNA binding site.

E22. The method of embodiment E21, wherein the miRNA binding site bindsto miR-122.

E23. The method of embodiment E21 or E22, wherein the miRNA binding sitebinds to miR-122-3p or miR-122-5p.

E24. The method of embodiment E23, wherein the miRNA binding sitecomprises a nucleotide sequence at least 80%, at least 85%, at least90%, at least 95%, or 100% identical to SEQ ID NO: 26, wherein the miRNAbinding site binds to miR-122.

E25. The method of embodiment E23, wherein the miRNA binding sitecomprises a nucleotide sequence at least 80%, at least 85%, at least90%, at least 95%, or 100% identical to SEQ ID NO: 24, wherein the miRNAbinding site binds to miR-122.

E26. The method of any one of embodiments E21 to E25, wherein thenucleotide sequence binds to SEQ ID NO: 22.

E27. The method of any one of embodiments E1 to E26, wherein thepolynucleotide further comprises a 5′ UTR.

E28. The method of embodiment E27, wherein the 5′ UTR comprises anucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence listed in Table 3.

E29. The method of any one of embodiments E1 to E28 which furthercomprises a 3′ UTR.

E30. The method of embodiment E29, wherein the 3′ UTR comprises anucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence listed in Table 4A or 4B.

E31. The method of embodiment E29 or E30, wherein the miRNA binding siteis inserted within the 3′ UTR.

E32. The method of embodiment E31, wherein the polynucleotide furthercomprises a spacer sequence between the open reading frame and the miRNAbinding site.

E33. The method of embodiment E32, wherein the spacer sequence comprisesat least about 10 nucleotides, at least about 20 nucleotides, at leastabout 30 nucleotides, at least about 40 nucleotides, at least about 50nucleotides, at least about 60 nucleotides, at least about 70nucleotides, at least about 80 nucleotides, at least about 90nucleotides, or at least about 100 nucleotides.

E34. The method of any one of embodiments E1 to E33, wherein thepolynucleotide further comprises a 5′ terminal cap.

E35. The method of embodiment E34, wherein the 5′ terminal cap is aCap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

E36. The method of any one of embodiments E1 to E35, wherein thepolynucleotide further comprises a 3′ polyA tail.

E37. The method of any one of embodiments E21 to E36, wherein thepolynucleotide comprises at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten miRNA binding sites.

E38. The methods of any one of embodiments E1 to E37, wherein thepolynucleotide is codon optimized.

E39. The method of any one of embodiments E1 to E38, wherein thepolynucleotide is in vitro transcribed (IVT).

E40. The method of any one of embodiments E1 to E38, wherein thepolynucleotide is chimeric.

E41. The method of any one of embodiments E1 to E38, wherein thepolynucleotide is circular.

E42. The method of any one of embodiments E1 to E41, wherein the OX40Lpolypeptide is fused to one or more heterologous polypeptides.

E43. The method of embodiment E42, wherein the one or more heterologouspolypeptides increase a pharmacokinetic property of the OX40Lpolypeptide.

E44. The method of any one of embodiments E1 to E43, wherein thepolynucleotide further comprises a second open reading frame encoding asecond polypeptide.

E45. The method of any one of embodiments E1 to E43, further comprisingadministering a second polynucleotide.

E46. The method of embodiment E45, wherein the second polynucleotidecomprises a second open reading frame encoding a second polypeptide.

E47. The method of any one of embodiments E44 to E46, wherein the secondopen reading frame is a second mRNA.

E48. The method of embodiment E47, wherein the second mRNA comprises atleast one chemically modified nucleoside.

E49. The method of any one of embodiments E1 to E48, wherein thepolynucleotide is formulated with a delivery agent.

E50. The method of embodiment E49, wherein the delivery agent comprisesa lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymericcompound, a peptide, a protein, a cell, a nanoparticle mimic, ananotube, or a conjugate.

E51. The method of embodiment E50, wherein the delivery agent is a lipidnanoparticle.

E52. The method of embodiment E51, wherein the lipid nanoparticlecomprises the lipid selected from the group consisting of DLin-DMA,DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA,PEG, PEG-DMG, PEGylated lipids, amino alcohol lipids, KL22, andcombinations thereof.

E53. The method of any one of embodiments E1 to E52, wherein thepolynucleotide is formulated for in vivo delivery.

E54. The method of embodiment E53, wherein the polynucleotide isformulated for subcutaneous, intravenous, intraperitoneal, intratumoral,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial,intraventricular, oral, inhalation spray, topical, rectal, nasal,buccal, vaginal, intratumoral, or implanted reservoir intramuscular,subcutaneous, intratumoral, or intradermal delivery.

E55. The method of any one of embodiments E1 to E53, wherein thepolynucleotide is administered subcutaneously, intravenously,intraperitoneally, intratumorally, intramuscularly, intra-articularly,intra-synovially, intrasternally, intrathecally, intrahepatically,intralesionally, intracranially, intraventricularly, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir.

E56. The method of any one of embodiments E1 to E55, wherein theadministration treats a cancer.

E57. The method of embodiment E56, wherein the cancer is selected fromthe group consisting of adrenal cortical cancer, advanced cancer, analcancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer,bone metastasis, brain tumors, brain cancer, breast cancer, childhoodcancer, cancer of unknown primary origin, Castleman disease, cervicalcancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewingfamily of tumors, eye cancer, gallbladder cancer, gastrointestinalcarcinoid tumors, gastrointestinal stromal tumors, gestationaltrophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cellcarcinoma, laryngeal and hypopharyngeal cancer, acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyeloid leukemia, chronic myelomonocytic leukemia, liver cancer,hepatocellular carcinoma (HCC), non-small cell lung cancer, small celllung cancer, lung carcinoid tumor, lymphoma of the skin, malignantmesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma in adult soft tissue, basal and squamouscell skin cancer, melanoma, small intestine cancer, stomach cancer,testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,Wilms tumor, secondary cancers caused by cancer treatment, and anycombination thereof.

E58. The method of any one of embodiments E1 to E57, wherein thepolynucleotide is delivered by a device comprising a pump, patch, drugreservoir, short needle device, single needle device, multiple needledevice, micro-needle device, jet injection device, ballisticpowder/particle delivery device, catheter, lumen, cryoprobe, cannula,microcanular, or devices utilizing heat, RF energy, electric current, orany combination thereof.

E59. The method of any one of embodiments E1 to E58, wherein thepolynucleotide is administered intratumorally to the subject at a unitdose of at least about 10 μg, at least about 12.5 μg, or at least about15 μg.

E60. The method of any one of embodiments E1 to E58, wherein theeffective amount is between about 0.10 μg/kg and about 1000 mg/kg.

E61. The method of any one of embodiments E6 to E60, wherein the numberof NK cells is increased at least five-fold at 24 hours after theadministration.

E62. The method of any one of embodiments E1 to E61, wherein the tumorgrowth is inhibited at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, or about 100% after the administration compared to acontrol polynucleotide that does not encode an OX40L polypeptide.

E63. The method of any one of embodiments E1 to E62, further comprisingadministering a PD-1 antagonist to the subject.

E64. The method of embodiment E63, wherein the PD-1 antagonist is anantibody or antigen binding portion thereof that specifically binds toPD-1.

E65. The method of embodiment E63 or E64, wherein the PD-1 antagonist isa monoclonal antibody.

E66. The method of any one of embodiments E63 to E65, wherein the PD-1antagonist is selected from the group consisting of Nivolumab,Pembrolizumab, Pidilizumab, and any combination thereof.

E67. The method of any one of embodiments E1 to E62, further comprisingadministering a PD-L1 antagonist to the subject.

E68. The method of embodiment E67, wherein the PD-L1 antagonist is anantibody that binds to PD-L1.

E69. The method of embodiment E67 or E68, wherein the PD-L1 antagonistis a monoclonal antibody.

E70. The method of any one of embodiments E67 to E69, wherein the PD-L1antagonist is selected from the group consisting of Durvalumab,Avelumab, MEDI473, BMS-936559, Atezolizumab, and any combinationthereof.

E71. The method of any one of embodiments E1 to E62, further comprisingadministering a CTLA-4 antagonist to the subject.

E72. The method of embodiment E71, wherein the CTLA-4 antagonist is anantibody or antigen binding portion thereof that specifically binds toCTLA-4.

E73. The method of embodiment E71 or E72, wherein the CTLA-4 antagonistis a monoclonal antibody.

E74. The method of any one of embodiments E71 to E73, wherein the CTLA-4antagonist is selected from the group consisting of Ipilimumab,Tremelimumab, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows an example of an OX40L encoding polynucleotide (mRNA). ThemRNA can comprise a 5′cap, 5′ UTR, an OFR (mRNA) encoding an OX40Lpolypeptide, a 3′UTR, a miR122 binding site, and a poly-A tail.

FIG. 2 shows expression of OX40L on the surface of B16F10 cells aftertreatment with a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. The left peaks represent the control (either mock-treatedor treated with negative control mRNA (non-translatable version of thesame mRNA containing multiple stop codons)). The right four peaksrepresent OX40L expression from the administration of 6.3 ng, 12.5 ng,25 ng, or 50 ng OX40L mRNA.

FIG. 3A shows expression of OX40L on the surface of HeLa cells aftertreatment with a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide; treatment was in the absence of mitomycin C. FIG. 3B showexpression of OX40L on the surface of MC-38 cells after treatment with apolynucleotide comprising an mRNA encoding an OX40L polypeptide;treatment was in the absence of mitomycin C. Peak 1 in FIGS. 3A and 3Bshows surface expression on mock treated cells. Peaks 2-6 show surfaceexpression on days 1, 2, 3, 5, and 7 (respectively) after treatment witha polynucleotide comprising an mRNA encoding an OX40L polypeptide.

FIG. 3C shows expression of OX40L on the surface of HeLa cells aftertreatment with a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide; treatment was in the presence of mitomycin C. FIG. 3D showsexpression of OX40L on the surface of MC-38 cells after treatment with apolynucleotide comprising an mRNA encoding an OX40L polypeptide;treatment was in the presence of mitomycin C. Peak 1 in FIGS. 3C and 3Dshows surface expression on mock treated cells. Peaks 2-6 show surfaceexpression on days 1, 2, 3, 5, and 7 (respectively) after treatment witha polynucleotide comprising an mRNA encoding an OX40L polypeptide.

FIG. 3E shows expression of human OX40L on the surface of HeLa cellsafter treatment with a polynucleotide comprising an mRNA encoding anOX40L polypeptide. Peak 1 shows the surface expression on mock treatedcells. Peaks 2-6 show surface expression on day 1, 2, 3, 4, and 5(respectively) after treatment with a polynucleotide comprising an mRNAencoding an OX40L polypeptide.

FIG. 3F shows quantitation of mouse OX40L protein in cell lysate andcell culture supernatant after treatment of HeLa cells with apolynucleotide comprising an mRNA encoding an OX40L polypeptide. FIG. 3Gshows quantitation of human OX40L protein in cell lysate and cellculture supernatant after treatment of HeLa cells with a polynucleotidecomprising an mRNA encoding an OX40L polypeptide. The y-axis in FIGS. 3Fand 3G shows the amount of protein as nanograms (ng) per well.

FIG. 4A shows a schematic drawing of the T-cell activation assay.OX40L-expressing B16F10 cells or HeLa cells were co-cultured with CD4⁺T-cells and anti-mouse CD3 antibody (B16F10 cells) or anti-human CD3antibody and soluble anti-human CD28 (HeLa cells). IL-2 production wasmeasured using ELISA as a correlate of T-cell activation. FIG. 4B showsresults of the T-cell activation assay as measured by mouse IL-2. FIG.4C shows results of the T-cell activation assay as measured by humanIL-2. The y-axis shows mIL2 expression in ng/ml.

FIG. 4D shows the data from FIG. 4C with schematic diagram showing theaddition of OX40L expressing cells to the naïve T-cell activation assay.

FIG. 4E shows a T-cell activation assay using pre-stimulated T-cellscultured in the presence or absence of OX40L expressing HeLa cells andin the presence or absence of anti-human CD3 antibody.

FIG. 5 shows luciferase flux levels in tumor tissue compared to livertissue in animals treated with a polynucleotide comprising an mRNAencoding a luciferase polypeptide. Representative symbols are asfollows. The open inverted triangle, open star, open diamond, shadeddiamond, and open circle show the luciferase flux in tumor tissue afteradministration of 50 μg, 25 μg, 12.5 μg, 6.25 μg, and 3.125 μg ofmOX40L_miR122 mRNA (respectively). The shaded inverted triangle, shadedstar, open square, shaded square, and open triangle show the luciferaseflux in liver tissue after administration of 50 μg, 25 μg, 12.5 μg, 6.25μg, and 3.125 μg of mOX40L_miR122 mRNA (respectively). The shaded circleand shaded triangle show luciferase flux in tumor tissue (shaded circle)and liver tissue (shaded triangle) after administration of PBS control.

FIG. 6 shows the amount of OX40L polypeptide present in melanoma tumortissue in animals treated with a polynucleotide comprising an mRNAencoding an OX40L polypeptide. The left panel shows 8 hours aftertreatment, and the right panel shows 24 hours after treatment.

FIG. 7A shows the amount of OX40L polypeptide present in colonadenocarcinoma tumor tissue in animals treated with a polynucleotidecomprising an mRNA encoding an OX40L polypeptide, a polynucleotidecomprising an mRNA encoding a NST-OX40L (non translatable OX40L mRNA),or no treatment. The OX40L expression was measured at 3 hours, 6 hours,24 hours, 48 hours, 72 hours, and 168 hours. FIG. 7B shows the amount ofOX40L polypeptide (upper) and mRNA (lower) present in tumor tissuefollowing administration of increasing doses of a polynucleotidecomprising an mRNA encoding an OX40L polypeptide. FIG. 7C shows theamount of OX40L polypeptide (upper) and mRNA (lower) present in livertissue following administration of the same polynucleotide. FIG. 7Dshows the amount of OX40L polypeptide (upper) and mRNA (lower) presentin spleen tissue following administration of the same polynucleotide.

FIG. 8A-8C show the in vivo efficacy (as measured at Day 42) ofadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide in a colon adenocarcinoma model. FIG. 8A shows tumor growthfor animals treated with a control mRNA (NT OX40L_miR122 control). FIG.8B shows tumor growth for animals treated with a polynucleotidecomprising an mRNA encoding an OX40L polypeptide (OX40L_miR122). FIG. 8Cshows a Kaplan-Meier survival curve for all treatment groups.(OX40L_miR122, NST_OX40L_miR122, and PBS).

FIG. 9A-9C show OX40L expression in A20 B-cell lymphoma tumors inanimals treated with a polynucleotide comprising an mRNA encoding anOX40L polypeptide. FIG. 9A shows OX40L expression quantitated innanograms per gram of tumor tissue, as measured by ELISA. FIG. 9B showsOX40L expression on the cell surface of tumor cells, as measured by flowcytometry. FIG. 9C shows OX40L expression on the cell surface of tumorcells as measured by flow cytometry.

FIG. 10A-10B show Natural Killer (NK) cell infiltration into the tumormicroenvironment in animals treated with a polynucleotide comprising anmRNA encoding an OX40L polypeptide. FIG. 10A shows the averagepercentage of live NK cells present in the tumor microenvironment. Theleft bar shows the percentage of the NK cells increased afteradministration of mOX40L_mRNA. The right bar shows the percentage of theNK cells increased after administration of NST mOX40L_mRNA. FIG. 10Bshows individual animal data from the same study.

FIG. 11A-11D show in vivo efficacy of administering a polynucleotidecomprising an mRNA encoding an OX40L polypeptide in a B-cell lymphomatumor model. FIG. 11A shows tumor growth in animals treated with acontrol mRNA (NST-FIX control). FIG. 11B shows tumor growth in animalstreated with a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide (OX40L_miR122). FIG. 11C shows the average tumor volume foreach group, as measured at Day 35. FIG. 11D shows Kaplan-Meier survivalcurves for each treatment group. The squares show the tumor volume afteradministration of OX40L_miR122. The triangles show the tumor volumeafter administration of NST-FIX (control).

FIG. 12A. shows in vivo immune response after administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Micewere inoculated with MC-38 colon adenocarcinoma cells. Once the tumorsreached palpable size, mice were administered a polynucleotidecomprising an mRNA encoding an OX40L polypeptide (OX40L_122; triangle),a control nonsense mRNA (NST-OX40L_122; inverted triangle), or PBS(square). Sixty days following administration of the polypeptide, micewere re-challenged with a second MC-38 tumor cell inoculation. FIG. 12Ashows the individual animal tumor during the first period through Day60. FIG. 12B shows the number of animals presenting with tumor growth 23days after re-challenge.

FIG. 13 shows OX40L expression in A20 tumors at various timepoints aftera first and/or second dose of a polynucleotide comprising an mRNAencoding an OX40L polypeptide. Expression is shown at 24 hours, 72hours, 7 days, and 14 days after administration of a first dose of thepolynucleotide and 24 hours, 72 hours, and 7 days after administrationof a second dose of the polynucleotide.

FIG. 14A-14C show different cell types present in the tumormicroenvironment following administration of a polynucleotide comprisingan mRNA encoding an OX40L polypeptide. FIG. 14A shows the percentage ofOX40L-expressing cells in A20 tumors that are cancer cells, immunecells, non-cancer/non-immune cells, and cells of myeloid lineage. FIG.14B shows the percentage of OX40L-expressing cells in MC38 tumors thatare tumor cells, immune cells, and cells of myeloid lineage. FIG. 14Cshows the percentage of myeloid cells in the tumor microenvironment thatare OX40L-expressing cells following administration of thepolynucleotide.

FIG. 15A-15D show the different types of immune cells that infiltratethe tumor microenvironment in A20 tumors following administration of apolynucleotide comprising an mRNA encoding an OX40L polypeptide. FIG.15A shows the percentage of NK cells in the tumor infiltrate 24 hoursafter treatment, as detected by the DX5 marker. FIG. 15B shows thepercentage of CD4⁺ T-cells in the tumor infiltrate 14 days aftertreatment, as detected by the CD4 marker. FIG. 15C shows the percentageof CD8⁺ T-cells in the tumor infiltrate 14 days after treatment, asdetected by the CD8 marker. FIG. 15D shows the percentage of CD8⁺T-cells in the tumor infiltrate of MC38 tumors 24 and 72 hours after afirst and second dose of a polynucleotide comprising an mRNA encoding anOX40L polypeptide.

FIG. 16A-16B show in vivo efficacy of administration of a polynucleotidecomprising an mRNA encoding an OX40L polypeptide in A20 tumors. FIG. 16Ashows tumor volume (measured in mm³) over time. Treatments are shown asfollows: mOX40L_miR122 (filled circles); control mRNA (NST) (opensquares); PBS (open triangles); and untreated (open circles). FIG. 16Bshows a Kaplan-Meier survival curve for the same animals.

FIG. 17A-17B show expression of OX40L protein in primary humanhepatocytes, human liver cancer cells (Hep3B), and human cervicalcarcinoma cells (HeLa) at 6 hours, 24 hours, and 48 hourspost-transfection. FIG. 17A shows expression of human OX40L polypeptideas measured in nanograms per well. FIG. 17B shows expression of mouseOX40L polypeptide as measured in nanograms per well.

FIG. 18A-18E show in vivo anti-tumor efficacy of mOX40L_miR122 deliveredintratumorally or intravenously. FIG. 18A shows tumor growth in animalstreated intratumorally with control mRNA (“NST-OX40L”) (arrows markinjection days). FIG. 18B shows tumor growth in animals treatedintratumorally with mOX40L_miR122 mRNA (“OX40L-miR122”) (arrows markinjection days). FIG. 18C shows tumor growth in animals treatedintravenously with control mRNA (“NST-OX40L”) (arrows mark injectiondays). FIG. 18D shows tumor growth in animals treated intravenously withmOX40L_miR122 mRNA (“OX40L-miR122”) (arrows mark injection days). FIG.18E shows tumor growth in animals treated intravenously with PBS (arrowsmark injection days).

FIG. 19 shows survival curves for animals treated intravenously withPBS, negative control mRNA (“NST-OX40L”), or mOX40L-miR122 mRNA(“OX40L”). Dose days are indicated by arrows.

FIG. 20A-20F show in vivo anti-tumor efficacy of combination therapycomprising a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide and an anti-PD-1 antibody. FIG. 20A shows tumor growth inanimals treated with intratumoral injections of control mRNA(“NST_mOX40L_122”) and control antibody (“Rat IgG2a”). FIG. 20B showstumor growth in animals treated with intratumoral injections ofmOX40L_miR122 (“mOX40L_122”) and control antibody (“Rat IgG2a”). FIG.20C shows tumor growth in animals treated with intratumoral injectionsof control mRNA (“NST_mOX40L_122”) and anti-PD-1 antibody (“anti-PD-1”).FIG. 20D shows tumor growth in animals treated with intratumoralinjections of mOX40L_miR122 (“mOX40L_122”) and anti-PD-1 antibody(“anti-PD-1”). FIG. 20E shows tumor growth in animals treated withintratumoral injections of anti-PD-1 antibody and PBS. FIG. 20F showstumor growth in animals treated with PBS and control antibody (“RatIgG2a”). CR=complete responder.

FIG. 21 shows survival curves for animals treated intratumorally withcombination therapy comprising control mRNA and control antibody(“NST_mOX40L_122+Rat IgG2a”), mOX40L_miR122 and control antibody(“mOX40L_122+Rat IgG2a”), control mRNA and anti-PD-1 antibody(“NST_mOX40L_122+anti-PD-1”), mOX40L_miR122 and anti-PD-1 antibody(“mOX40L_122+anti-PD-1”), anti-PD-1 antibody and PBS (“PBS+anti-PD-1”),and PBS and control antibody (“PBS+Rat IgG2a”).

FIG. 22A-22B show a memory immune response in animals treated withcombination therapy comprising a polynucleotide comprising an mRNAencoding an OX40L polypeptide and a miR122 binding site and an anti-PD-1antibody. Animals were initially treated with intratumoral injections ofmOX40L_miR122 and anti-PD-1 antibody as shown in FIG. 20D. Four animalsidentified as complete responders (CR) were re-challenged with MC38tumor cells. FIG. 22A shows individual tumor growth in naïve animalschallenged with MC38 tumor cells. FIG. 22B shows individual tumor growthin the four CR animals re-challenged with MC38 tumor cells.

DETAILED DESCRIPTION

The present application is directed to methods of activating T cells orincreasing the number of Natural Killer (NK) cells in a subject using apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Thepolynucleotide described herein can further reduce or decrease a size ofa tumor or inhibit a tumor growth in a subject in need thereof byproviding a polynucleotide comprising an mRNA which encodes an OX40Lpolypeptide.

The headings provided herein are not limitations of the various aspectsor aspects of the disclosure, which can be defined by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety. Before describing the present disclosure in detail, it is tobe understood that this disclosure is not limited to specificcompositions or process steps, as such can vary.

I. DEFINITIONS

In order that the present disclosure can be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The disclosure includes embodiments in which exactly one member of thegroup is present in, employed in, or otherwise relevant to a givenproduct or process. The disclosure includes embodiments in which morethan one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process.

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein. Incertain aspects, the term “a” or “an” means “single.” In other aspects,the term “a” or “an” includes “two or more” or “multiple.”

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within thedisclosure. Where a value is explicitly recited, it is to be understoodthat values which are about the same quantity or amount as the recitedvalue are also within the scope of the disclosure. Where a combinationis disclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the disclosure.Conversely, where different elements or groups of elements areindividually disclosed, combinations thereof are also disclosed. Whereany element of a disclosure is disclosed as having a plurality ofalternatives, examples of that disclosure in which each alternative isexcluded singly or in any combination with the other alternatives arealso hereby disclosed; more than one element of a disclosure can havesuch exclusions, and all combinations of elements having such exclusionsare hereby disclosed.

Nucleotides are referred to by their commonly accepted single-lettercodes. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation. Nucleotides are referred to herein bytheir commonly known one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Accordingly, A represents adenine,C represents cytosine, G represents guanine, T represents thymine, and Urepresents uracil.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Unless otherwise indicated, aminoacid sequences are written left to right in amino to carboxyorientation.

About: The term “about” as used in connection with a numerical valuethroughout the specification and the claims denotes an interval ofaccuracy, familiar and acceptable to a person skilled in the art. Ingeneral, such interval of accuracy is +10%.

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the disclosure, to the tenthof the unit of the lower limit of the range, unless the context clearlydictates otherwise.

Amino acid substitution: The term “amino acid substitution” refers toreplacing an amino acid residue present in a parent sequence (e.g., aconsensus sequence) with another amino acid residue. An amino acid canbe substituted in a parent sequence, for example, via chemical peptidesynthesis or through recombinant methods known in the art. Accordingly,a reference to a “substitution at position X” refers to the substitutionof an amino acid present at position X with an alternative amino acidresidue. In some aspects, substitution patterns can be describedaccording to the schema AnY, wherein A is the single letter codecorresponding to the amino acid naturally present at position n, and Yis the substituting amino acid residue. In other aspects, substitutionpatterns can be described according to the schema An(YZ), wherein A isthe single letter code corresponding to the amino acid residuesubstituting the amino acid naturally present at position X, and Y and Zare alternative substituting amino acid residue, i.e., In the context ofthe present disclosure, substitutions (even when they referred to asamino acid substitution) are conducted at the nucleic acid level, i.e.,substituting an amino acid residue with an alternative amino acidresidue is conducted by substituting the codon encoding the first aminoacid with a codon encoding the second amino acid.

Conservative amino acid substitution: A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, or histidine),acidic side chains (e.g., aspartic acid or glutamic acid), unchargedpolar side chains (e.g., glycine, asparagine, glutamine, serine,threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine, ortryptophan), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, or histidine). Thus, if an amino acid in a polypeptide isreplaced with another amino acid from the same side chain family, theamino acid substitution is considered to be conservative. In anotheraspect, a string of amino acids can be conservatively replaced with astructurally similar string that differs in order and/or composition ofside chain family members.

Non-conservative amino acid substitutions include those in which (i) aresidue having an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) acysteine or proline is substituted for, or by, any other residue, or(iv) a residue having a bulky hydrophobic or aromatic side chain (e.g.,Val, His, Ile or Trp) is substituted for, or by, one having a smallerside chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

Other amino acid substitutions can be readily identified by workers ofordinary skill. For example, for the amino acid alanine, a substitutioncan be taken from any one of D-alanine, glycine, beta-alanine,L-cysteine and D-cysteine. For lysine, a replacement can be any one ofD-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine,ornithine, or D-ornithine. Generally, substitutions in functionallyimportant regions that can be expected to induce changes in theproperties of isolated polypeptides are those in which (i) a polarresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, oralanine; (ii) a cysteine residue is substituted for (or by) any otherresidue; (iii) a residue having an electropositive side chain, e.g.,lysine, arginine or histidine, is substituted for (or by) a residuehaving an electronegative side chain, e.g., glutamic acid or asparticacid; or (iv) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having such a side chain, e.g.,glycine. The likelihood that one of the foregoing non-conservativesubstitutions can alter functional properties of the protein is alsocorrelated to the position of the substitution with respect tofunctionally important regions of the protein: some non-conservativesubstitutions can accordingly have little or no effect on biologicalproperties.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats a tumor, an effectiveamount of an agent is, for example, an amount sufficient to reduce ordecrease a size of a tumor or to inhibit a tumor growth, as compared tothe response obtained without administration of the agent. The term“effective amount” can be used interchangeably with “effective dose,”“therapeutically effective amount,” or “therapeutically effective dose.”

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the disclosure, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the disclosure, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.For example, the percent identity between two nucleotide sequences canbe determined using the algorithm of Meyers and Miller (CABIOS, 1989,4:11-17), which has been incorporated into the ALIGN program (version2.0) using a PAM120 weight residue table, a gap length penalty of 12 anda gap penalty of 4. The percent identity between two nucleotidesequences can, alternatively, be determined using the GAP program in theGCG software package using an NWSgapdna.CMP matrix. Methods commonlyemployed to determine percent identity between sequences include, butare not limited to those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48:1073 (1988). Techniques for determining identity arecodified in publicly available computer programs. Exemplary computersoftware to determine homology between two sequences include, but arenot limited to, GCG program package, Devereux, J., et al., Nucleic AcidsResearch, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F.et al., J. Molec. Biol., 215, 403 (1990)).

Immune response: The term “immune response” refers to the action of, forexample, lymphocytes, antigen presenting cells, phagocytic cells,granulocytes, and soluble macromolecules produced by the above cells orthe liver (including antibodies, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances (e.g., nucleotide sequence or proteinsequence) can have varying levels of purity in reference to thesubstances from which they have been associated. Isolated substancesand/or entities can be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated. In some embodiments, isolated agents are more than about80%, about 85%, about 90%, a, at 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof.

A polynucleotide, vector, polypeptide, cell, or any compositiondisclosed herein which is “isolated” is a polynucleotide, vector,polypeptide, cell, or composition which is in a form not found innature. Isolated polynucleotides, vectors, polypeptides, or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some aspects, apolynucleotide, vector, polypeptide, or composition which is isolated issubstantially pure.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Polynucleotide: The term “polynucleotide” as used herein refers topolymers of nucleotides of any length, including ribonucleotides,deoxyribonucleotides, analogs thereof, or mixtures thereof. This termrefers to the primary structure of the molecule. Thus, the term includestriple-, double- and single-stranded deoxyribonucleic acid (“DNA”), aswell as triple-, double- and single-stranded ribonucleic acid (“RNA”).It also includes modified, for example by alkylation, and/or by capping,and unmodified forms of the polynucleotide. More particularly, the term“polynucleotide” includes polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), includingtRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, anyother type of polynucleotide which is an N- or C-glycoside of a purineor pyrimidine base, and other polymers containing normucleotidicbackbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”)and polymorpholino polymers, and other synthetic sequence-specificnucleic acid polymers providing that the polymers contain nucleobases ina configuration which allows for base pairing and base stacking, such asis found in DNA and RNA. In particular aspects, the polynucleotidecomprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. Insome aspects, the synthetic mRNA comprises at least one unnaturalnucleobase. In some aspects, all nucleobases of a certain class havebeen replaced with unnatural nucleobases (e.g., all uridines in apolynucleotide disclosed herein can be replaced with an unnaturalnucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide(e.g., a synthetic RNA or a synthetic DNA) comprises only naturalnucleobases, i.e., A, C, T and U in the case of a synthetic DNA, or A,C, T, and U in the case of a synthetic RNA.

The skilled artisan will appreciate that the T bases in the codon mapsdisclosed herein are present in DNA, whereas the T bases would bereplaced by U bases in corresponding RNAs. For example, acodon-nucleotide sequence disclosed herein in DNA form, e.g., a vectoror an in-vitro translation (IVT) template, would have its T basestranscribed as U based in its corresponding transcribed mRNA. In thisrespect, both codon-optimized DNA sequences (comprising T) and theircorresponding RNA sequences (comprising U) are consideredcodon-optimized nucleotide sequence of the present disclosure. A skilledartisan would also understand that equivalent codon-maps can begenerated by replaced one or more bases with non-natural bases. Thus,e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map),which in turn would correspond to a ΨΨC codon (RNA map in which U hasbeen replaced with pseudouridine).

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine (U.S. Pat.No. 5,681,702 to Collins et al.). Isocytosine is available from SigmaChemical Co. (St. Louis, Mo.); isocytidine can be prepared by the methoddescribed by Switzer et al. (1993) Biochemistry 32:10489-10496 andreferences cited therein; 2′-deoxy-5-methyl-isocytidine can be preparedby the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 andreferences cited therein; and isoguanine nucleotides can be preparedusing the method described by Switzer et al., 1993, supra, and Mantschet al., 1993, Biochem. 14:5593-5601, or by the method described in U.S.Pat. No. 5,780,610 to Collins et al. Other nonnatural base pairs can besynthesized by the method described in Piccirilli et al., 1990, Nature343:33-37, for the synthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modifiednucleotide units which form unique base pairs are known, such as thosedescribed in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 andSwitzer et al., supra.

Nucleic acid sequence: The terms “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide” are used interchangeably and refer to acontiguous nucleic acid sequence. The sequence can be either singlestranded or double stranded DNA or RNA, e.g., an mRNA.

The phrase “nucleotide sequence encoding” and variants thereof refers tothe nucleic acid (e.g., an mRNA or DNA molecule) coding sequence thatcomprises a nucleotide sequence which encodes a polypeptide orfunctional fragment thereof as set forth herein. The coding sequence canfurther include initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of an individual or mammalto which the nucleic acid is administered. The coding sequence canfurther include sequences that encode signal peptides.

Polypeptide: The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can comprise modified amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids such as homocysteine, ornithine, p-acetylphenylalanine,D-amino acids, and creatine), as well as other modifications known inthe art.

The term, as used herein, refers to proteins, polypeptides, and peptidesof any size, structure, or function. Polypeptides include gene products,naturally occurring polypeptides, synthetic polypeptides, homologs,orthologs, paralogs, fragments and other equivalents, variants, andanalogs of the foregoing. A polypeptide can be a single OX40Lpolypeptide or can be a multi-molecular complex such as a dimer, trimeror tetramer. They can also comprise single chain or multichainpolypeptides. Most commonly disulfide linkages are found in multichainpolypeptides. The term polypeptide can also apply to amino acid polymersin which one or more amino acid residues are an artificial chemicalanalogue of a corresponding naturally occurring amino acid.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure takento maintain health and prevent the spread of disease. An “immuneprophylaxis” refers to a measure to produce active or passive immunityto prevent the spread of disease.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Signal transduction pathway: A “signal transduction pathway” refers tothe biochemical relationship between a variety of signal transductionmolecules that play a role in the transmission of a signal from oneportion of a cell to another portion of a cell. As used herein, thephrase “cell surface receptor” includes, for example, molecules andcomplexes of molecules capable of receiving a signal and thetransmission of such a signal across the plasma membrane of a cell. Anexample of a “cell surface receptor” of the present disclosure is theOX40 receptor.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Subject: By “subject” or “individual” or “animal” or “patient” or“mammal,” is meant any subject, particularly a mammalian subject, forwhom diagnosis, prognosis, or therapy is desired. Mammalian subjectsinclude, but are not limited to, humans, domestic animals, farm animals,zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, cows; primates such as apes,monkeys, orangutans, and chimpanzees; canids such as dogs and wolves;felids such as cats, lions, and tigers; equids such as horses, donkeys,and zebras; bears, food animals such as cows, pigs, and sheep; ungulatessuch as deer and giraffes; rodents such as mice, rats, hamsters andguinea pigs; and so on. In certain embodiments, the mammal is a humansubject. In other embodiments, a subject is a human patient. In aparticular embodiment, a subject is a human patient in need of a cancertreatment.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Treating, treatment, therapy: As used herein, the term “treating” or“treatment” or “therapy” refers to partially or completely alleviating,ameliorating, improving, relieving, delaying onset of, inhibitingprogression of, reducing severity of, and/or reducing incidence of oneor more symptoms or features of a hyper-proliferative disease, e.g.,cancer. For example, “treating” cancer can refer to inhibiting survival,growth, and/or spread of a tumor. Treatment can be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition and/or to a subject who exhibits only early signs of adisease, disorder, and/or condition for the purpose of decreasing therisk of developing pathology associated with the disease, disorder,and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified can,but does not always, refer to the wild type or native form of abiomolecule. Molecules can undergo a series of modifications wherebyeach modified molecule can serve as the “unmodified” starting moleculefor a subsequent modification.

II. METHODS OF USE

The methods of the present disclosure provide for the use of apolynucleotide comprising an mRNA encoding an OX40L polypeptide.

Human OX40L was first identified on the surface of human lymphocytesinfected with human T-cell leukemia virus type-I (HTLV-I) by Tanaka etal. (Tanaka et al., International Journal of Cancer (1985),36(5):549-55). Human OX40L is a 34 kDa glycosylated type IItransmembrane protein that exists on the surface of cells as a trimer.OX40L comprises a cytoplasmic domain (amino acids 1-23), a transmembranedomain (amino acids 24-50) and an extracellular domain (amino acids51-183). OX40L is also referred to as Tumor Necrosis Factor Superfamily(ligand) Member 4 (TNFSF4), CD252, CD134L, Tax-TranscriptionallyActivated Glycoprotein 1 (TXGP1), Glycoprotein 34 (GP34), and ACT-4-L.

Thus, the present disclosure provides a method of activating T cells ina subject in need thereof comprising administering to the subject apolynucleotide comprising an mRNA encoding an OX40L polypeptide. In oneaspect, the activation of T cells in the subject is directed to ananti-tumor immune response in the subject. In another aspect, theactivated T cells in the subject reduce or decrease the size of a tumoror inhibit the growth of a tumor in the subject. Activation of T cellscan be measured using applications in the art such as measuring T cellproliferation; measuring cytokine production with enzyme-linkedimmunosorbant assays (ELISA) or enzyme-linked immunospot assays(ELISPOT); or detection of cell-surface markers associated with T cellactivation (e.g., CD69, CD40L, CD137, CD25, CD71, CD26, CD27, CD28,CD30, CD154, and CD134) with techniques such as flow cytometry.

In some embodiments, the present disclosure provides a method ofinducing T cell proliferation in a subject in need thereof comprisingadministering to the subject a polynucleotide comprising an mRNAencoding an OX40L polypeptide. In one aspect, the T cell proliferationin the subject is directed to an anti-tumor immune response in thesubject. In another aspect, the T cell proliferation in the subjectreduces or decreases the size of a tumor or inhibits the growth of atumor in the subject. T cell proliferation can be measured usingapplications in the art such as cell counting, viability staining,optical density assays, or detection of cell-surface markers associatedwith T cell activation (e.g., CD69, CD40L, CD137, CD25, CD71, CD26,CD27, CD28, CD30, CD154, and CD134) with techniques such as flowcytometry.

In other embodiments, the present disclosure provides a method ofinducing T cell infiltration in a tumor of a subject in need thereofcomprising administering to the subject a polynucleotide comprising anmRNA encoding an OX40L polypeptide. In one aspect, the T cellinfiltration in a tumor of the subject is directed to an anti-tumorimmune response in the subject. In another aspect, the T cellinfiltration in a tumor of the subject reduces or decreases the size ofa tumor or inhibits the growth of a tumor in the subject. T cellinfiltration in a tumor can be measured using applications in the artsuch as tissue sectioning and staining for cell markers, measuring localcytokine production at the tumor site, or detection of T cell-surfacemarkers with techniques such as flow cytometry.

In other embodiments, the present disclosure provides a method ofinducing a memory T cell response in a subject in need thereofcomprising administering to the subject a polynucleotide comprising anmRNA encoding an OX40L polypeptide. In one aspect, the memory T cellresponse in the subject is directed to an anti-tumor immune response inthe subject. In another aspect, the memory T cell response in thesubject reduces or decreases the size of a tumor or inhibits the growthof a tumor in the subject. A memory T cell response can be measuredusing applications in the art such as measuring T cell markersassociated with memory T cells, measuring local cytokine productionrelated to memory immune response, or detecting memory T cell-surfacemarkers with techniques such as flow cytometry.

In certain embodiments, the activated T cells by the present methods areCD4⁺ cells, CD8⁺ cells, CD62⁺ (L-selectin⁺) cells, CD69⁺ cells, CD40L⁺cells, CD137⁺ cells, CD25⁺ cells, CD71⁺ cells, CD26⁺ cells, CD27⁺ cells,CD28⁺ cells, CD30⁺ cells, CD45⁺ cells, CD45RA⁺ cells, CD45RO⁺ cells,CD11b⁺ cells, CD154⁺ cells, CD134⁺ cells, CXCR3⁺ cells, CCR4⁺ cells,CCR6⁺ cells, CCR7⁺ cells, CXCR5⁺ cells, Crth2⁺ cells, gamma delta Tcells, or any combination thereof. In some embodiments, the activated Tcells by the present methods are Th₁ cells. In other embodiments, the Tcells activated by the present methods are Th₂ cells. In otherembodiments, the T cells activated by the present disclosure arecytotoxic T cells.

In some embodiments, the infiltrating T cells by the present methods areCD4⁺ cells, CD8⁺ cells, CD62⁺ (L-selectin⁺) cells, CD69⁺ cells, CD40L⁺cells, CD137⁺ cells, CD25⁺ cells, CD71⁺ cells, CD26⁺ cells, CD27⁺ cells,CD28⁺ cells, CD30⁺ cells, CD45⁺ cells, CD45RA⁺ cells, CD45RO⁺ cells,CD11b⁺ cells, CD154⁺ cells, CD134⁺ cells, CXCR3⁺ cells, CCR4⁺ cells,CCR6⁺ cells, CCR7⁺ cells, CXCR5⁺ cells, Crth2⁺ cells, gamma delta Tcells, or any combination thereof. In some embodiments, the infiltratingT cells by the present methods are Th₁ cells. In other embodiments, theinfiltrating T cells by the present methods are Th₂ cells. In otherembodiments, the infiltrating T cells by the present disclosure arecytotoxic T cells.

In some embodiments, the memory T cells induced by the present methodsare CD4⁺ cells, CD8⁺ cells, CD62⁺ (L-selectin⁺) cells, CD69⁺ cells,CD40L⁺ cells, CD137⁺ cells, CD25⁺ cells, CD71⁺ cells, CD26⁺ cells, CD27⁺cells, CD28⁺ cells, CD30⁺ cells, CD45⁺ cells, CD45RA⁺ cells, CD45RO⁺cells, CD11b⁺ cells, CD154⁺ cells, CD134⁺ cells, CXCR3⁺ cells, CCR4⁺cells, CCR6⁺ cells, CCR7⁺ cells, CXCR5⁺ cells, Crth2⁺ cells, gamma deltaT cells, or any combination thereof. In some embodiments, the memory Tcells by the present methods are Th₁ cells. In other embodiments, thememory T cells by the present methods are Th₂ cells. In otherembodiments, the memory T cells by the present disclosure are cytotoxicT cells.

The present disclosure further provides a method of increasing thenumber of Natural Killer (NK) cells in a subject in need thereofcomprising administering a polynucleotide comprising an mRNA encoding anOX40L polypeptide. In one aspect, the increase in the number of NK cellsin the subject is directed to an anti-tumor immune response in thesubject. In another aspect, the increase in the number of NK cells inthe subject reduces or decreases the size of a tumor or inhibits thegrowth of a tumor in the subject. Increases in the number of NK cells ina subject can be measured using applications in the art such asdetection of NK cell-surface markers (e.g., CD335/NKp46; CD336/NKp44;CD337/NPp30) or intracellular NK cell markers (e.g., perforin;granzymes; granulysin).

In certain embodiments, administration of the mRNA encoding an OX40Lpolypeptide increases the total number of NK cells in the subjectcompared to the number of NK cells in a subject who is not administeredwith the mRNA encoding an OX40L polypeptide. In other embodiments,administration of the mRNA encoding an OX40L polypeptide increases thetotal number of NK cells in the subject compared to a subject who isadministered a dendritic cell transduced with the mRNA encoding an OX40Lpolypeptide. In other embodiments, administration of the mRNA encodingan OX40L polypeptide increases the number of NK cells in the subjectwithin the tumor microenvironment compared to that of a subject who isnot administered with the mRNA encoding the OX40L polypeptide. In otherembodiments, administration of the mRNA encoding an OX40L polypeptideincreases the number of NK cells in a subject within the tumormicroenvironment compared to that of a subject who is administered adendritic cell transduced with the mRNA encoding an OX40L polypeptide.In other embodiments, the concentration of NK cells within the tumormicroenvironment is increased while the total number of NK cells in thesubject remains the same.

In certain embodiments of the disclosure, the number of NK cells isincreased at least about two-fold, at least about three-fold, at leastabout four-fold, at least about five-fold, at least about six-fold, atleast about seven-fold, at least about eight-fold, at least aboutnine-fold, or at least about ten-fold compared to a control (e.g.,saline or an mRNA without OX40L expression). In a particular embodiment,the number of NK cells is increased by the mRNA encoding an OX40Lpolypeptide at least about two-fold compared to a control (e.g., salineor an mRNA without OX40L expression).

The present disclosure further provides a method of increasing IL-2 in asubject in need thereof comprising administering a polynucleotidecomprising an mRNA encoding an OX40L polypeptide to increase IL-2 in thesubject in need thereof. In one aspect, the increase in IL-2 in thesubject is directed to an anti-tumor immune response in the subject. Inone embodiment, the increase in IL-2 expression by the polynucleotidecomprising an mRNA encoding an OX40L polypeptide is at least abouttwo-fold, at least about three-fold, at least about four-fold, at leastabout five-fold, or at least about six-fold higher than a control (e.g.,PBS treated). The IL-2 expression can be measured using any availabletechniques, such as ELISA or ELISPOT assays.

In other aspects, the present disclosure provides a method of increasingIL-4 in a subject in need thereof comprising administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide toincrease IL-4 in the subject in need thereof. In one aspect, theincrease in IL-4 in the subject is directed to an anti-tumor immuneresponse in the subject. In one embodiment, the increase in IL-4expression by the polynucleotide comprising an mRNA encoding an OX40Lpolypeptide is at least about two-fold, at least about three-fold, atleast about four-fold, at least about five-fold, or at least aboutsix-fold higher than a control (e.g., PBS treated). The IL-4 expressioncan be measured using any available techniques, such as ELISA or ELISPOTassays.

In other aspects, the present disclosure provides a method of increasingIL-21 in a subject in need thereof comprising administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide toincrease IL-21 in the subject in need thereof. In one aspect, theincrease in IL-21 in the subject is directed to an anti-tumor immuneresponse in the subject. In one embodiment, the increase in IL-21expression by the polynucleotide comprising an mRNA encoding an OX40Lpolypeptide is at least about two-fold, at least about three-fold, atleast about four-fold, at least about five-fold, or at least aboutsix-fold higher than a control (e.g., PBS treated). The IL-21 expressioncan be measured using any available techniques, such as ELISA or ELISPOTassays.

The polynucleotide (e.g., mRNA) of the present disclosure can beadministered in any route available, including, but not limited to,intratumoral, enteral, gastroenteral, epidural, oral, transdermal,epidural (peridural), intracerebral (into the cerebrum),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intraperitoneal (into the peritoneum),intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In other embodiments, themRNA of the present disclosure is administered parenterally (e.g.,includes subcutaneous, intravenous, intraperitoneal, intratumoral,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques), intraventricularly, orally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir.

The present disclosure also includes a method of activating T cells in asubject in need thereof comprising intratumorally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Incertain embodiments, the intratumoral administration of the present mRNAcan increase the efficacy of the anti-tumor effect (e.g., T cellactivation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellproliferation in a subject in need thereof comprising intratumorallyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intratumoral administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., T cell proliferation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellinfiltration in a tumor in a subject in need thereof comprisingintratumorally administering a polynucleotide comprising an mRNAencoding an OX40L polypeptide. In certain embodiments, the intratumoraladministration of the present mRNA can increase the efficacy of theanti-tumor effect (e.g., T cell infiltrationin a tumor) compared toother routes of administration.

The present disclosure also includes a method of inducing a memory Tcells response in a subject in need thereof comprising intratumorallyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intratumoral administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., memory T cell response) compared to other routes ofadministration.

The present disclosure also includes a method of increasing the numberof NK cells in a subject in need thereof comprising intratumorallyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intratumoral administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., NK cell increase) compared to other routes of administration. ThemRNA of the present disclosure can be formulated specifically for theintratumoral delivery as shown elsewhere herein.

The present disclosure further provides a method of increasing IL-2 in asubject in need thereof comprising intratumorally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-2 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-4 in asubject in need thereof comprising intratumorally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-4 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-21 ina subject in need thereof comprising intratumorally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-21 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure also includes a method of activating T cells in asubject in need thereof comprising intraperitoneally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Incertain embodiments, the intraperitoneal administration of the presentmRNA can increase the efficacy of the anti-tumor effect (e.g., T cellactivation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellproliferation in a subject in need thereof comprising intraperitoneallyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intraperitoneal administrationof the present mRNA can increase the efficacy of the anti-tumor effect(e.g., T cell proliferation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellinfiltration in a tumor in a subject in need thereof comprisingintraperitoneally administering a polynucleotide comprising an mRNAencoding an OX40L polypeptide. In certain embodiments, theintraperitoneal administration of the present mRNA can increase theefficacy of the anti-tumor effect (e.g., T cell infiltration in a tumor)compared to other routes of administration.

The present disclosure also includes a method of inducing a memory Tcells response in a subject in need thereof comprising intraperitoneallyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intraperitoneal administrationof the present mRNA can increase the efficacy of the anti-tumor effect(e.g., memory T cell response) compared to other routes ofadministration.

The present disclosure also includes a method of increasing the numberof NK cells in a subject in need thereof comprising intraperitonealadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intraperitoneal administrationof the present mRNA can increase the efficacy of the anti-tumor effect(e.g., NK cell increase) compared to other routes of administration. ThemRNA of the present disclosure can be formulated specifically for theintraperitoneal delivery as shown elsewhere herein.

The present disclosure further provides a method of increasing IL-2 in asubject in need thereof comprising intraperitoneally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-2 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-4 in asubject in need thereof comprising intraperitoneally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-4 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-21 ina subject in need thereof comprising intraperitoneally administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-21 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure also includes a method of activating T cells in asubject in need thereof comprising intravenously administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide. Incertain embodiments, the intravenous administration of the present mRNAcan increase the efficacy of the anti-tumor effect (e.g., T cellactivation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellproliferation in a subject in need thereof comprising intravenouslyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intravenous administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., T cell proliferation) compared to other routes of administration.

The present disclosure also includes a method of inducing T cellinfiltration in a tumor in a subject in need thereof comprisingintravenously administering a polynucleotide comprising an mRNA encodingan OX40L polypeptide. In certain embodiments, the intravenousadministration of the present mRNA can increase the efficacy of theanti-tumor effect (e.g., T cell infiltration in a tumor) compared toother routes of administration.

The present disclosure also includes a method of inducing a memory Tcell response in a subject in need thereof comprising intravenouslyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intravenous administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., memory T cell response) compared to other routes ofadministration.

The present disclosure also includes a method of increasing the numberof NK cells in a subject in need thereof comprising intravenouslyadministering a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide. In certain embodiments, the intravenous administration ofthe present mRNA can increase the efficacy of the anti-tumor effect(e.g., NK cell increase) compared to other routes of administration. ThemRNA of the present disclosure can be formulated specifically for theintravenous delivery as shown elsewhere herein.

The present disclosure further provides a method of increasing IL-2 in asubject in need thereof comprising intravenously administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-2 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-4 in asubject in need thereof comprising intravenously administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-4 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further provides a method of increasing IL-21 ina subject in need thereof comprising intravenously administering apolynucleotide comprising an mRNA encoding an OX40L polypeptide to thesubject. In one aspect, the increase in IL-21 in the subject is directedto an anti-tumor immune response in the subject.

The present disclosure further includes a method of activating T cellsin a subject in need thereof comprising administering (e.g.,intratumorally, intraperitoneally, or intravenously) a polynucleotidecomprising an mRNA encoding an OX40L polypeptide as a monotherapy, i.e.,without any other anti-cancer agent in combination. The presentdisclosure also provides a method of increasing the number of NaturalKiller (NK) cells in a subject in need thereof comprising administering(e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide as amonotherapy. Some embodiments of the disclosure also include a method ofincreasing IL-2, IL-4, IL-21, or any combination thereof in a subject inneed thereof comprising administering (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide as a monotherapy.

In certain embodiments, the disclosure is directed to a method ofactivating T cells in a subject in need thereof comprising administering(e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with one or more anti-cancer agents to the subject. In otherembodiments, the disclosure includes a method of increasing the numberof NK cells in a subject in need thereof comprising administering (e.g.,intratumorally, intraperitoneally, or intravenously) a polynucleotidecomprising an mRNA encoding an OX40L polypeptide in combination with oneor more anti-cancer agents to the subject. Other embodiments of thedisclosure also provides a method of increasing IL-2, IL-4, IL-21, orany combination thereof in a subject in need thereof comprisingadministering (e.g., intratumorally, intraperitoneally, orintravenously) a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide with one or more anti-cancer agents to the subject. In someembodiments, the one or more anti-cancer agents are an mRNA. In certainembodiments, the one or more anti-cancer agents are an mRNA encoding atumor antigen. In other embodiments, the one or more anti-cancer agentsare not a tumor antigen or an mRNA encoding a tumor antigen.

In some embodiments, the one or more anti-cancer agents are an approvedagent by the United States Food and Drug Administration. In otherembodiments, the one or more anti-cancer agents are a pre-approved agentby the United States Food and Drug Administration.

In some aspects, the subject for the present methods has been treatedwith one or more standard of care therapies. In other aspects, thesubject for the present methods has not been responsive to one or morestandard of care therapies or anti-cancer therapies. In one aspect, thesubject has been previously treated with a PD-1 antagonist prior to thepolynucleotide of the present disclosure. In another aspect, the subjecthas been treated with a monoclonal antibody that binds to PD-1 prior tothe polynucleotide of the present disclosure. In another aspect, thesubject has been treated with an anti-PD-1 monoclonal antibody therapyprior to the polynucleotide of the present methods. In other aspects,the anti-PD-1 monoclonal antibody therapy comprises Nivolumab,Pembrolizumab, Pidilizumab, or any combination thereof. In anotheraspect, the subject has been treated with a monoclonal antibody thatbinds to PD-L1 prior to the polynucleotide of the present disclosure. Inanother aspect, the subject has been treated with an anti-PD-L1monoclonal antibody therapy prior to the polynucleotide of the presentmethods. In other aspects, the anti-PD-L1 monoclonal antibody therapycomprises Durvalumab, Avelumab, MEDI473, BMS-936559, Atezolizumab, orany combination thereof.

In some aspects, the subject has been treated with a CTLA-4 antagonistprior to the polynucleotide of the present disclosure. In anotheraspect, the subject has been previously treated with a monoclonalantibody that binds to CTLA-4 prior to the polynucleotide of the presentdisclosure. In another aspect, the subject has been treated with ananti-CTLA-4 monoclonal antibody prior to the polynucleotide of thepresent disclosure. In other aspects, the anti-CTLA-4 antibody therapycomprises Ipilimumab or Tremelimumab.

In some aspects, the disclosure is directed to a method of activating Tcells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with a PD-1 antagonist. In another aspect, the disclosure isdirected to a method of activating T cells in a subject in need thereofcomprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an antibody orantigen-binding portion thereof that specifically binds to PD-1. Inanother aspect, the disclosure is directed to a method of activating Tcells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with an anti-PD-1 monoclonal antibody.

In one embodiment, the anti-PD-1 antibody (or an antigen-binding portionthereof) useful for the disclosure is pembrolizumab. Pembrolizumab (alsoknown as “KEYTRUDA®”, lambrolizumab, and MK-3475) is a humanizedmonoclonal IgG4 antibody directed against human cell surface receptorPD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab isdescribed, for example, in U.S. Pat. No. 8,900,587; see alsohttp://www.cancer.gov/drugdictionary?cdrid=695789 (last accessed: Dec.14, 2014). Pembrolizumab has been approved by the FDA for the treatmentof relapsed or refractory melanoma and advanced NSCLC.

In another embodiment, the anti-PD-1 antibody useful for the disclosureis nivolumab. Nivolumab (also known as “OPDIVO®”; formerly designated5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P)PD-1 immune checkpoint inhibitor antibody that selectively preventsinteraction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking thedown-regulation of antitumor T-cell functions (U.S. Pat. No. 8,008,449;Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). Nivolumab has shownactivity in a variety of advanced solid tumors including renal cellcarcinoma (renal adenocarcinoma, or hypernephroma), melanoma, andnon-small cell lung cancer (NSCLC) (Topalian et al., 2012a; Topalian etal., 2014; Drake et al., 2013; WO 2013/173223.

In other embodiments, the anti-PD-1 antibody is MEDI0680 (formerlyAMP-514), which is a monoclonal antibody against the PD-1 receptor.MEDI0680 is described, for example, in U.S. Pat. No. 8,609,089B2 or inhttp://www.cancer.gov/drugdictionary?cdrid=756047 (last accessed Dec.14, 2014).

In certain embodiments, the anti-PD-1 antibody is BGB-A317, which is ahumanized monoclonal antibody. BGB-A317 is described in U.S. Publ. No.2015/0079109.

In certain embodiments, a PD-1 antagonist is AMP-224, which is a B7-DCFc fusion protein. AMP-224 is discussed in U.S. Publ. No. 2013/0017199or inhttp://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595(last accessed Jul. 8, 2015).

In other embodiments, the disclosure includes a method of increasing thenumber of NK cells in a subject in need thereof comprising administeringto the subject (e.g., intratumorally, intraperitoneally, orintravenously) a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide in combination with a PD-1 antagonist. In another aspect,the disclosure is directed to a method of increasing the number of NKcells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with an antibody or antigen-binding portion thereof thatspecifically binds to PD-1. In another aspect, the disclosure isdirected to a method of increasing the number of NK cells in a subjectin need thereof comprising administering to the subject (e.g.,intratumorally, intraperitoneally, or intravenously) a polynucleotidecomprising an mRNA encoding an OX40L polypeptide in combination with ananti-PD-1 monoclonal antibody. In other aspects, the anti-PD-1monoclonal antibody comprises Nivolumab, Pembrolizumab, Pidilizumab, orany combination thereof.

In other embodiments, the disclosure includes a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with a PD-1 antagonist. Inanother aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an antibody orantigen-binding portion thereof that specifically binds to PD-1. Inanother aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an anti-PD-1monoclonal antibody. In other aspects, the anti-PD-1 monoclonal antibodycomprises Nivolumab, Pembrolizumab, Pidilizumab, or any combinationthereof.

In another aspect, the disclosure is directed to a method of activatingT cells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with an antibody or antigen-binding portion thereof thatspecifically binds to PD-L1. In another aspect, the disclosure isdirected to a method of activating T cells in a subject in need thereofcomprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an anti-PD-L1monoclonal antibody. In other aspects, the anti-PD-L1 monoclonalantibody comprises Durvalumab, Avelumab, MED1473, BMS-936559,Atezolizumab, or any combination thereof.

In certain embodiments, the anti-PD-L1 antibody useful for thedisclosure is MSB0010718C (also called Avelumab; See US 2014/0341917) orBMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Pat. No.7,943,743; WO 2013/173223). In other embodiments, the anti-PD-L1antibody is MPDL3280A (also known as RG7446) (see, e.g., Herbst et al.(2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Pat. No. 8,217,149),MEDI4736 (also called Durvalumab; Khleif (2013) In: Proceedings from theEuropean Cancer Congress 2013; Sep. 27-Oct. 1, 2013; Amsterdam, TheNetherlands.

In another aspect, the disclosure is directed to a method of increasingthe number of NK cells in a subject in need thereof comprisingadministering to the subject (e.g., intratumorally, intraperitoneally,or intravenously) a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide in combination with an antibody or antigen-binding portionthereof that specifically binds to PD-L1. In another aspect, thedisclosure is directed to a method of increasing the number of NK cellsin a subject in need thereof comprising administering to the subject(e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with an anti-PD-L1 monoclonal antibody. In other aspects,the anti-PD-L1 monoclonal antibody comprises Durvalumab, Avelumab,MEDI473, BMS-936559, Atezolizumab, or any combination thereof.

In another aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an antibody orantigen-binding portion thereof that specifically binds to PD-L1. Inanother aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an anti-PD-L1monoclonal antibody. In other aspects, the anti-PD-L1 monoclonalantibody comprises Durvalumab, Avelumab, MEDI473, BMS-936559,Atezolizumab, or any combination thereof.

In some aspects, the disclosure is directed to a method of activating Tcells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with a CTLA-4 antagonist. In another aspect, the disclosureis directed to a method of activating T cells in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an antibody orantigen-binding portion thereof that specifically binds to CTLA-4. Inanother aspect, the disclosure is directed to a method of activating Tcells in a subject in need thereof comprising administering to thesubject (e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with an anti-CTLA-4 monoclonal antibody. In other aspects,the anti-CTLA-4 monoclonal antibody comprises Ipilimumab orTremelimumab, or any combination thereof. In some aspects, thedisclosure is directed to a method of increasing the number of NK cellsin a subject in need thereof comprising administering to the subject(e.g., intratumorally, intraperitoneally, or intravenously) apolynucleotide comprising an mRNA encoding an OX40L polypeptide incombination with a CTLA-4 antagonist. In another aspect, the disclosureis directed to a method of increasing the number of NK cells in asubject in need thereof comprising administering to the subject (e.g.,intratumorally, intraperitoneally, or intravenously) a polynucleotidecomprising an mRNA encoding an OX40L polypeptide in combination with anantibody or antigen-binding portion thereof that specifically binds toCTLA-4. In another aspect, the disclosure is directed to a method ofincreasing the number of NK cells in a subject in need thereofcomprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an anti-CTLA-4monoclonal antibody. In other aspects, the anti-CTLA-4 monoclonalantibody comprises Ipilimumab or Tremelimumab, or any combinationthereof.

In some aspects, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with a CTLA-4 antagonist.In another aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an antibody orantigen-binding portion thereof that specifically binds to CTLA-4. Inanother aspect, the disclosure is directed to a method of increasingIL-2, IL-4, IL-21, or any combination thereof in a subject in needthereof comprising administering to the subject (e.g., intratumorally,intraperitoneally, or intravenously) a polynucleotide comprising an mRNAencoding an OX40L polypeptide in combination with an anti-CTLA-4monoclonal antibody.

An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (nowknown as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat.No. 6,984,720. Another anti-CTLA-4 antibody useful for the presentmethods is tremelimumab (also known as CP-675,206). Tremelimumab ishuman IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described inWO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648A2.

Thus, the administration of mRNA as referred to in the presentdisclosure is not in the form of a dendritic cell comprising an mRNAencoding an OX40L protein. Rather, the administration in the presentdisclosure is a direct administration of the mRNA encoding an OX40Lpolypeptide to the subject (e.g., to a tumor in a subject).

Diseases, Disorders and/or Conditions

In some embodiments, the polynucleotides (e.g., mRNA) encoding an OX40Lpolypeptide of the present disclosure can be used to reduce or decreasea size of a tumor or inhibit a tumor growth in a subject in needthereof.

In some embodiments, the tumor is associated with a disease, disorder,and/or condition. In a particular embodiment, the disease, disorder,and/or condition is a cancer. Thus, in one aspect, the administration ofthe polynucleotide (e.g., mRNA) encoding an OX40L polypeptide treats acancer.

A “cancer” refers to a broad group of various diseases characterized bythe uncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth results in the formation of malignant tumors thatinvade neighboring tissues and can also metastasize to distant parts ofthe body through the lymphatic system or bloodstream. A “cancer” or“cancer tissue” can include a tumor at various stages. In certainembodiments, the cancer or tumor is stage 0, such that, e.g., the canceror tumor is very early in development and has not metastasized. In someembodiments, the cancer or tumor is stage I, such that, e.g., the canceror tumor is relatively small in size, has not spread into nearby tissue,and has not metastasized. In other embodiments, the cancer or tumor isstage II or stage III, such that, e.g., the cancer or tumor is largerthan in stage 0 or stage I, and it has grown into neighboring tissuesbut it has not metastasized, except potentially to the lymph nodes. Inother embodiments, the cancer or tumor is stage IV, such that, e.g., thecancer or tumor has metastasized. Stage IV can also be referred to asadvanced or metastatic cancer.

In some aspects, the cancer can include, but is not limited to, adrenalcortical cancer, advanced cancer, anal cancer, aplastic anemia, bileductcancer, bladder cancer, bone cancer, bone metastasis, brain tumors,brain cancer, breast cancer, childhood cancer, cancer of unknown primaryorigin, Castleman disease, cervical cancer, colon/rectal cancer,endometrial cancer, esophagus cancer, Ewing family of tumors, eyecancer, gallbladder cancer, gastrointestinal carcinoid tumors,gastrointestinal stromal tumors, gestational trophoblastic disease,Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal andhypopharyngeal cancer, acute lymphocytic leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia,chronic myelomonocytic leukemia, liver cancer, non-small cell lungcancer, small cell lung cancer, lung carcinoid tumor, lymphoma of theskin, malignant mesothelioma, multiple myeloma, myelodysplasticsyndrome, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumors, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue,basal and squamous cell skin cancer, melanoma, small intestine cancer,stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroidcancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrommacroglobulinemia, Wilms tumor and secondary cancers caused by cancertreatment.

In some aspects, the tumor is a solid tumor. A “solid tumor” includes,but is not limited to, sarcoma, melanoma, carcinoma, or other solidtumor cancer. “Sarcoma” refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include, but are not limited to, chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acra-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma,metastatic melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, e.g., acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidernoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum.

Additional cancers that can be treated include, e.g., Leukemia,Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrinecancer, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, adrenal corticalcancer, prostate cancer, Müllerian cancer, ovarian cancer, peritonealcancer, fallopian tube cancer, or uterine papillary serous carcinoma.

III. COMPOSITIONS Polynucleotides

The polynucleotide of the present application comprises an mRNA encodingan OX40L polypeptide. OX40L is the ligand for OX40 (CD134). OX40L hasalso been designated CD252 (cluster of differentiation 252), tumornecrosis factor (ligand) superfamily, member 4, tax-transcriptionallyactivated glycoprotein 1, TXGP1, or gp34.

Human OX40L is 183 amino acids in length and contains three domains: acytoplasmic domain of amino acids 1-23; a transmembrane domain of aminoacids 24-50, and an extracellular domain of amino acids 51-183.

In some embodiments, the polynucleotide comprises an mRNA encoding amammalian OX40L polypeptide. In some embodiments, the mammalian OX40Lpolypeptide is a murine OX40L polypeptide. In some embodiments, themammalian OX40L polypeptide is a human OX40L polypeptide. In someembodiments, the OX40L polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 1. In another embodiment, the OX40L polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the OX40L polypeptide comprises an amino acidsequence at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% identicalto an amino acid sequence listed in Table 1 (e.g., selected from SEQ IDNOs: 1-3) or an amino acid sequence encoded by a nucleotide sequencelisted in Table 1, wherein the amino acid sequence is capable of bindingto an OX40 receptor.

In other embodiments, the OX40L polypeptide useful for the disclosurecomprises an amino acid sequence listed in Table 1 with one or moreconservative substitutions, wherein the conservative substitutions donot affect the binding of the OX40L polypeptide to an OX40 receptor,i.e., the amino acid sequence binds to the OX40 receptor after thesubstitutions.

In certain embodiments, the OX40L polypeptide comprises an amino acidsequence at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% identicalto an extracellular domain of OX40L (e.g., SEQ ID NO: 2), wherein theOX40L polypeptide binds to an OX40 receptor.

In other embodiments, a nucleotide sequence (i.e., mRNA) encoding anOX40L polypeptide comprises a sequence at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or 100% identical to a nucleic acid sequence listed in Table1 (e.g., selected from SEQ ID NOs: 4-21).

TABLE 1 OX40LPolypeptide and Polynucleotide Sequences EncodedDescription Sequence SEQ ID NO: OX40L Amino acid sequenceMERVQPLEENVGNAARPRFERNKLLLVASVIQG SEQ ID NO: 1 (TNFSF4)of tumor necrosis LGLLLCFTYICLHFSALQVSHRYPRIQSIKVQF 183 aa factor ligandTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDG superfamily member 4FYLISLKGYFSQEVNISLHYQKDEEPLFQLKKV isoform 1 [Homo sapiens]RSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHV NP_003317 NGGELILIHQNPGEFCVL OX40LAmino acid sequence MVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED SEQ ID NO: 2(TNFSF4) of tumor necrosis EIMKVQNNSVIINCDGFYLISLKGYFSQEVNIS 133 aafactor ligand LHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKV superfamily member 4YLNVTTDNTSLDDFHVNGGELILIHQNPGEFCV isoform 2 [Homo sapiens] LNP_001284491 OX40L Amino acid sequence MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGSEQ ID NO: 3 (TNFSF4) of tumor necrosisIKGAGMLLCFIYVCLQLSSSPAKDPPIQRLRGA 198 aa factor ligandVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCD superfamily member 4GLYIIYLKGSFFQEVKIDLHFREDHNPISIPML [Mus musculus]NDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEH NP_033478LQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL OX40L Nucleotide sequence ofAUGGAAAGGGUCCAACCCCUGGAAGAGAAUGUG SEQ ID NO: 4 (TNFSF4)TNFSF4 tumor necrosis GGAAAUGCAGCCAGGCCAAGAUUCGAGAGGAAC 549 ntsfactor (ligand) AAGCUAUUGCUGGUGGCCUCUGUAAUUCAGGGA superfamily, member 4,CUGGGGCUGCUCCUGUGCUUCACCUACAUCUGC open reading frameCUGCACUUCUCUGCUCUUCAGGUAUCACAUCGG [Homo sapiens]UAUCCUCGAAUUCAAAGUAUCAAAGUACAAUUU ACCGAAUAUAAGAAGGAGAAAGGUUUCAUCCUCACUUCCCAAAAGGAGGAUGAAAUCAUGAAGGUG CAGAACAACUCAGUCAUCAUCAACUGUGAUGGGUUUUAUCUCAUCUCCCUGAAGGGCUACUUCUCC CAGGAAGUCAACAUUAGCCUUCAUUACCAGAAGGAUGAGGAGCCCCUCUUCCAACUGAAGAAGGUC AGGUCUGUCAACUCCUUGAUGGUGGCCUCUCUGACUUACAAAGACAAAGUCUACUUGAAUGUGACC ACUGACAAUACCUCCCUGGAUGACUUCCAUGUGAAUGGCGGAGAACUGAUUCUUAUCCAUCAAAAU CCUGGUGAAUUCUGUGUCCUU OX40LNucleotide sequence of GGCCCUGGGACCUUUGCCUAUUUUCUGAUUGAU SEQ ID NO: 5(TNFSF4) homo sapiens tumor AGGCUUUGUUUUGUCUUUACCUCCUUCUUUCUGnecrosis factor (ligand) GGGAAAACUUCAGUUUUAUCGCACGUUCCCCUUsuperfamily, member 4 UUCCAUAUCUUCAUCUUCCCUCUACCCAGAUUG(TNFSF4), transcript UGAAGAUGGAAAGGGUCCAACCCCUGGAAGAGA variant 1, mRNAAUGUGGGAAAUGCAGCCAGGCCAAGAUUCGAGA NM_003326GGAACAAGCUAUUGCUGGUGGCCUCUGUAAUUC AGGGACUGGGGCUGCUCCUGUGCUUCACCUACAUCUGCCUGCACUUCUCUGCUCUUCAGGUAUCAC AUCGGUAUCCUCGAAUUCAAAGUAUCAAAGUACAAUUUACCGAAUAUAAGAAGGAGAAAGGUUUCA UCCUCACUUCCCAAAAGGAGGAUGAAAUCAUGAAGGUGCAGAACAACUCAGUCAUCAUCAACUGUG AUGGGUUUUAUCUCAUCUCCCUGAAGGGCUACUUCUCCCAGGAAGUCAACAUUAGCCUUCAUUACC AGAAGGAUGAGGAGCCCCUCUUCCAACUGAAGAAGGUCAGGUCUGUCAACUCCUUGAUGGUGGCCU CUCUGACUUACAAAGACAAAGUCUACUUGAAUGUGACCACUGACAAUACCUCCCUGGAUGACUUCC AUGUGAAUGGCGGAGAACUGAUUCUUAUCCAUCAAAAUCCUGGUGAAUUCUGUGUCCUUUGAGGGG CUGAUGGCAAUAUCUAAAACCAGGCACCAGCAUGAACACCAAGCUGGGGGUGGACAGGGCAUGGAU UCUUCAUUGCAAGUGAAGGAGCCUCCCAGCUCAGCCACGUGGGAUGUGACAAGAAGCAGAUCCUGG CCCUCCCGCCCCCACCCCUCAGGGAUAUUUAAAACUUAUUUUAUAUACCAGUUAAUCUUAUUUAUC CUUAUAUUUUCUAAAUUGCCUAGCCGUCACACCCCAAGAUUGCCUUGAGCCUACUAGGCACCUUUG UGAGAAAGAAAAAAUAGAUGCCUCUUCUUCAAGAUGCAUUGUUUCUAUUGGUCAGGCAAUUGUCAU AAUAAACUUAUGUCAUUGAAAACGGUACCUGACUACCAUUUGCUGGAAAUUUGACAUGUGUGUGGC AUUAUCAAAAUGAAGAGGAGCAAGGAGUGAAGGAGUGGGGUUAUGAAUCUGCCAAAGGUGGUAUGA ACCAACCCCUGGAAGCCAAAGCGGCCUCUCCAAGGUUAAAUUGAUUGCAGUUUGCAUAUUGCCUAA AUUUAAACUUUCUCAUUUGGUGGGGGUUCAAAAGAAGAAUCAGCUUGUGAAAAAUCAGGACUUGAA GAGAGCCGUCUAAGAAAUACCACGUGCUUUUUUUCUUUACCAUUUUGCUUUCCCAGCCUCCAAACA UAGUUAAUAGAAAUUUCCCUUCAAAGAACUGUCUGGGGAUGUGAUGCUUUGAAAAAUCUAAUCAGU GACUUAAGAGAGAUUUUCUUGUAUACAGGGAGAGUGAGAUAACUUAUUGUGAAGGGUUAGCUUUAC UGUACAGGAUAGCAGGGAACUGGACAUCUCAGGGUAAAAGUCAGUACGGAUUUUAAUAGCCUGGGG AGGAAAACACAUUCUUUGCCACAGACAGGCAAAGCAACACAUGCUCAUCCUCCUGCCUAUGCUGAG AUACGCACUCAGCUCCAUGUCUUGUACACACAGAAACAUUGCUGGUUUCAAGAAAUGAGGUGAUCC UAUUAUCAAAUUCAAUCUGAUGUCAAAUAGCACUAAGAAGUUAUUGUGCCUUAUGAAAAAUAAUGA UCUCUGUCUAGAAAUACCAUAGACCAUAUAUAGUCUCACAUUGAUAAUUGAAACUAGAAGGGUCUA UAAUCAGCCUAUGCCAGGGCUUCAAUGGAAUAGUAUCCCCUUAUGUUUAGUUGAAAUGUCCCCUUA ACUUGAUAUAAUGUGUUAUGCUUAUGGCGCUGUGGACAAUCUGAUUUUUCAUGUCAACUUUCCAGA UGAUUUGUAACUUCUCUGUGCCAAACCUUUUAUAAACAUAAAUUUUUGAGAUAUGUAUUUUAAAAU UGUAGCACAUGUUUCCCUGACAUUUUCAAUAGAGGAUACAACAUCACAGAAUCUUUCUGGAUGAUU CUGUGUUAUCAAGGAAUUGUACUGUGCUACAAUUAUCUCUAGAAUCUCCAGAAAGGUGGAGGGCUG UUCGCCCUUACACUAAAUGGUCUCAGUUGGAUUUUUUUUUCCUGUUUUCUAUUUCCUCUUAAGUAC ACCUUCAACUAUAUUCCCAUCCCUCUAUUUUAAUCUGUUAUGAAGGAAGGUAAAUAAAAAUGCUAA AUAGAAGAAAUUGUAGGUAAGGUAAGAGGAAUCAAGUUCUGAGUGGCUGCCAAGGCACUCACAGAA UCAUAAUCAUGGCUAAAUAUUUAUGGAGGGCCUACUGUGGACCAGGCACUGGGCUAAAUACUUACA UUUACAAGAAUCAUUCUGAGACAGAUAUUCAAUGAUAUCUGGCUUCACUACUCAGAAGAUUGUGUG UGUGUUUGUGUGUGUGUGUGUGUGUGUAUUUCACUUUUUGUUAUUGACCAUGUUCUGCAAAAUUGC AGUUACUCAGUGAGUGAUAUCCGAAAAAGUAAACGUUUAUGACUAUAGGUAAUAUUUAAGAAAAUG CAUGGUUCAUUUUUAAGUUUGGAAUUUUUAUCUAUAUUUCUCACAGAUGUGCAGUGCACAUGCAGG CCUAAGUAUAUGUUGUGUGUGUUGUUUGUCUUUGAUGUCAUGGUCCCCUCUCUUAGGUGCUCACUC GCUUUGGGUGCACCUGGCCUGCUCUUCCCAUGUUGGCCUCUGCAACCACACAGGGAUAUUUCUGCU AUGCACCAGCCUCACUCCACCUUCCUUCCAUCAAAAAUAUGUGUGUGUGUCUCAGUCCCUGUAAGU CAUGUCCUUCACAGGGAGAAUUAACCCUUCGAUAUACAUGGCAGAGUUUUGUGGGAAAAGAAUUGA AUGAAAAGUCAGGAGAUCAGAAUUUUAAAUUUGACUUAGCCACUAACUAGCCAUGUAACCUUGGGA AAGUCAUUUCCCAUUUCUGGGUCUUGCUUUUCUUUCUGUUAAAUGAGAGGAAUGUUAAAUAUCUAA CAGUUUAGAAUCUUAUGCUUACAGUGUUAUCUGUGAAUGCACAUAUUAAAUGUCUAUGUUCUUGUU GCUAUGAGUCAAGGAGUGUAACCUUCUCCUUUACUAUGUUGAAUGUAUUUUUUUCUGGACAAGCUU ACAUCUUCCUCAGCCAUCUUUGUGAGUCCUUCAAGAGCAGUUAUCAAUUGUUAGUUAGAUAUUUUC UAUUUAGAGAAUGCUUAAGGGAUUCCAAUCCCGAUCCAAAUCAUAAUUUGUUCUUAAGUAUACUGG GCAGGUCCCCUAUUUUAAGUCAUAAUUUUGUAUUUAGUGCUUUCCUGGCUCUCAGAGAGUAUUAAU AUUGAUAUUAAUAAUAUAGUUAAUAGUAAUAUUGCUAUUUACAUGGAAACAAAUAAAAGAUCUCAG AAUUCACUAAAAAAAAAAA OX40LNucleotide sequence of AUUGCUUUUUGUCUCCUGUUCUGGGACCUUUAU SEQ ID NO: 6(TNFSF4) Mus musculus tumor CUUCUGACCCGCAGGCUUGACUUUGCCCUUAUU 1609 ntsnecrosis factor (ligand) GGCUCCUUUGUGGUGAAGAGCAGUCUUCCCCCAsuperfamily, member 4 GGUUCCCCGCCACAGCUGUAUCUCCUCUGCACC (Tnfsf4), mRNACCGACUGCAGAGAUGGAAGGGGAAGGGGUUCAA NM_009452CCCCUGGAUGAGAAUCUGGAAAACGGAUCAAGG CCAAGAUUCAAGUGGAAGAAGACGCUAAGGCUGGUGGUCUCUGGGAUCAAGGGAGCAGGGAUGCUU CUGUGCUUCAUCUAUGUCUGCCUGCAACUCUCUUCCUCUCCGGCAAAGGACCCUCCAAUCCAAAGA CUCAGAGGAGCAGUUACCAGAUGUGAGGAUGGGCAACUAUUCAUCAGCUCAUACAAGAAUGAGUAU CAAACUAUGGAGGUGCAGAACAAUUCGGUUGUCAUCAAGUGCGAUGGGCUUUAUAUCAUCUACCUG AAGGGCUCCUUUUUCCAGGAGGUCAAGAUUGACCUUCAUUUCCGGGAGGAUCAUAAUCCCAUCUCU AUUCCAAUGCUGAACGAUGGUCGAAGGAUUGUCUUCACUGUGGUGGCCUCUUUGGCUUUCAAAGAU AAAGUUUACCUGACUGUAAAUGCUCCUGAUACUCUCUGCGAACACCUCCAGAUAAAUGAUGGGGAG CUGAUUGUUGUCCAGCUAACGCCUGGAUACUGUGCUCCUGAAGGAUCUUACCACAGCACUGUGAAC CAAGUACCACUGUGAAUUCCACUCUGAGGGUGGACGGGACACAGGUUCUUUCUCGAGAGAGAUGAG UGCAUCCUGCUCAUGAGAUGUGACUGAAUGCAGAGCCUACCCUACUUCCUCACUCAGGGAUAUUUA AAUCAUGUCUUACAUAACAGUUGACCUCUCAUUCCCAGGAUUGCCUUGAGCCUGCUAAGAGCUGUU CUGGGAAUGAAAAAAAAAAUAAAUGUCUCUUCAAGACACAUUGCUUCUGUCGGUCAGAAGCUCAUC GUAAUAAACAUCUGCCACUGAAAAUGGCGCUUGAUUGCUAUCUUCUAGAAUUUUGAUGUUGUCAAA AGAAAGCAAAACAUGGAAAGGGUGGUGUCCACCGGCCAGUAGGAGCUGGAGUGCUCUCUUCAAGGU UAAGGUGAUAGAAGUUUACAUGUUGCCUAAAACUGUCUCUCAUCUCAUGGGGGGCUUGGAAAGAAG AUUACCCCGUGGAAAGCAGGACUUGAAGAUGACUGUUUAAGCAACAAGGUGCACUCUUUUCCUGGC CCCUGAAUACACAUAAAAGACAACUUCCUUCAAAGAACUACCUAGGGACUAUGAUACCCACCAAAG AACCACGUCAGCGAUGCAAAGAAAACCAGGAGAGCUUUGUUUAUUUUGCAGAGUAUACGAGAGAUU UUACCCUGAGGGCUAUUUUUAUUAUACAGGAUGAGAGUGAACUGGAUGUCUCAGGAUAAAGGCCAA GAAGGAUUUUUCACAGUCUGAGCAAGACUGUUUUUGUAGGUUCUCUCUCCAAAACUUUUAGGUAAA UUUUUGAUAAUUUUAAAAUUUUUAGUUAUAUUUUUGGACCAUUUUCAAUAGAAGAUUGAAACAUUU CCAGAUGGUUUCAUAUCCCCACAAG OX40LCodon-optimized AUGGAGAGAGUGCAGCCCCUGGAGGAGAACGUG SEQ ID NO: 7 (TNFSF4)sequence 1 for ENSP GGCAACGCCGCCAGACCCAGAUUCGAGAGAAAC 549 nts 281834AAGCUGCUGCUGGUGGCCAGCGUGAUCCAGGGC CUGGGCCUGCUGCUGUGCUUCACCUACAUCUGCCUGCACUUCAGCGCCCUGCAGGUGAGCCACAGA UACCCCAGAAUCCAGAGCAUCAAGGUGCAGUUCACCGAGUACAAGAAGGAGAAGGGCUUCAUCCUG ACCAGCCAGAAGGAGGACGAGAUCAUGAAGGUGCAGAACAACAGCGUGAUCAUCAACUGCGACGGC UUCUACCUGAUCAGCCUGAAGGGCUACUUCAGCCAGGAGGUGAACAUCAGCCUGCACUACCAGAAG GACGAGGAGCCCCUGUUCCAGCUGAAGAAGGUGAGAAGCGUGAACAGCCUGAUGGUGGCCAGCCUG ACCUACAAGGACAAGGUGUACCUGAACGUGACCACCGACAACACCAGCCUGGACGACUUCCACGUG AACGGCGGCGAGCUGAUCCUGAUCCACCAGAACCCCGGCGAGUUCUGCGUGCUG OX40L Codon-optimizedAUGGAGCGUGUGCAGCCUCUUGAGGAGAAUGUG SEQ ID NO: 8 (TNFSF4)sequence 2 for ENSP GGAAAUGCAGCCCGGCCUCGAUUCGAACGUAAU 549 nts 281834AAACUCCUGCUCGUGGCCUCCGUGAUCCAGGGU CUCGGUUUAUUGCUGUGUUUUACCUAUAUAUGCUUACACUUUAGUGCAUUACAGGUCUCACACCGG UACCCUCGCAUUCAGUCUAUAAAAGUGCAGUUUACCGAGUAUAAGAAGGAGAAAGGUUUUAUACUG ACUUCUCAGAAAGAGGACGAGAUCAUGAAGGUGCAGAAUAAUAGCGUCAUUAUCAACUGCGAUGGA UUCUAUCUAAUUUCCCUAAAGGGGUACUUCAGCCAGGAGGUCAAUAUAUCACUGCACUAUCAAAAG GACGAGGAGCCCCUGUUUCAACUGAAGAAAGUGCGAUCAGUUAACUCUCUGAUGGUUGCCUCUCUG ACCUAUAAGGACAAAGUCUACUUGAACGUGACAACUGACAACACCUCACUGGAUGACUUUCAUGUG AAUGGGGGGGAACUGAUUCUUAUCCAUCAGAAUCCAGGAGAAUUCUGUGUGCUC OX40L Codon-optimizedAUGGAGCGGGUGCAGCCCCUGGAGGAGAAUGUG SEQ ID NO: 9 (TNFSF4)sequence 3 for ENSP GGCAAUGCUGCCCGGCCCAGGUUUGAAAGAAAC 549 nts 281834AAGCUGCUGCUGGUGGCCAGCGUCAUCCAGGGC CUGGGCCUGCUGCUGUGCUUCACCUACAUCUGCCUGCACUUCAGCGCCCUGCAGGUGAGCCACCGC UACCCCCGCAUCCAGAGCAUCAAGGUGCAGUUCACAGAGUACAAGAAGGAGAAGGGCUUCAUCCUG ACCAGCCAGAAGGAGGAUGAGAUCAUGAAGGUGCAGAACAACAGCGUCAUCAUCAACUGUGAUGGC UUCUACCUGAUCAGCCUGAAGGGCUACUUCAGCCAGGAGGUGAACAUCAGCCUGCACUACCAGAAG GAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGCUCUGUGAACAGCCUGAUGGUGGCCAGCCUG ACCUACAAGGACAAGGUGUACCUGAAUGUGACCACAGACAACACCAGCCUGGAUGACUUCCACGUG AAUGGAGGAGAGCUGAUCCUGAUCCACCAGAACCCUGGAGAGUUCUGUGUGCUG OX40L Codon-optimizedAUGGAGCGGGUGCAGCCCCUGGAGGAGAACGUG SEQ ID NO: 10 (TNFSF4)sequence 4 for ENSP GGCAACGCCGCCCGCCCGCGUUUUGAGCGAAAU 549 nts 281834AAGUUACUGCUUGUUGCAUCUGUGAUACAGGGG UUGGGUUUACUUCUUUGCUUUACAUAUAUUUGUCUCCACUUUAGUGCGCUUCAGGUAUCCCAUCGG UACCCGCGCAUCCAGUCAAUCAAGGUCCAGUUCACUGAAUAUAAAAAGGAGAAAGGAUUCAUUCUG ACUUCACAAAAAGAGGACGAAAUCAUGAAAGUGCAGAACAACUCUGUAAUUAUAAACUGCGAUGGG UUCUAUCUGAUCAGUCUGAAGGGAUAUUUUAGCCAGGAAGUAAAUAUUUCACUACAUUAUCAGAAG GACGAAGAACCACUUUUUCAACUGAAGAAAGUCCGGUCCGUGAACUCCCUGAUGGUUGCUAGCCUU ACCUACAAGGAUAAAGUCUAUUUAAACGUCACAACAGAUAACACUAGCCUCGACGAUUUCCAUGUG AACGGAGGUGAACUGAUAUUGAUCCAUCAAAACCCCGGCGAGUUCUGCGUUUUA OX40L Codon-optimizedAUGGAGCGGGUCCAGCCCCUCGAGGAGAACGUU SEQ ID NO: 11 (TNFSF4)sequence 5 for ENSP GGUAAUGCCGCACGUCCCAGGUUUGAACGCAAC 549 nts 281834AAGCUGCUGUUGGUGGCCAGCGUCAUUCAGGGG CUGGGUUUGUUGCUGUGCUUCACUUACAUCUGUCUGCAUUUUAGUGCACUCCAGGUGUCCCACCGC UACCCCCGUAUCCAAUCCAUUAAAGUCCAAUUUACCGAAUACAAAAAAGAGAAGGGUUUCAUUCUU ACCUCCCAGAAGGAGGAUGAAAUUAUGAAGGUGCAGAACAAUUCUGUUAUCAUCAACUGUGACGGA UUCUAUCUGAUUUCACUGAAGGGAUACUUUUCCCAGGAGGUGAACAUCAGUCUGCAUUAUCAGAAG GACGAAGAACCGCUUUUUCAACUGAAGAAGGUUAGGAGUGUGAACUCCUUAAUGGUAGCCAGCCUG ACAUAUAAGGACAAGGUAUAUCUGAACGUCACCACUGAUAACACCUCUUUAGACGAUUUUCAUGUA AAUGGGGGAGAAUUGAUACUCAUUCACCAGAAUCCGGGUGAGUUUUGUGUUCUG OX40L Codon-optimizedAUGGUGAGCCACAGAUACCCCAGAAUCCAGAGC SEQ ID NO: 12 (TNFSF4)sequence 1 for ENSP AUCAAGGUGCAGUUCACCGAGUACAAGAAGGAG 399 nts 356691AAGGGCUUCAUCCUGACCAGCCAGAAGGAGGAC GAGAUCAUGAAGGUGCAGAACAACAGCGUGAUCAUCAACUGCGACGGCUUCUACCUGAUCAGCCUG AAGGGCUACUUCAGCCAGGAGGUGAACAUCAGCCUGCACUACCAGAAGGACGAGGAGCCCCUGUUC CAGCUGAAGAAGGUGAGAAGCGUGAACAGCCUGAUGGUGGCCAGCCUGACCUACAAGGACAAGGUG UACCUGAACGUGACCACCGACAACACCAGCCUGGACGACUUCCACGUGAACGGCGGCGAGCUGAUC CUGAUCCACCAGAACCCCGGCGAGUUCUGCGUG CUGOX40L Codon-optimized AUGGUUUCUCACCGUUACCCACGGAUCCAGUCU SEQ ID NO: 13(TNFSF4) sequence 2 for ENSP AUCAAGGUUCAGUUUACCGAGUACAAAAAGGAA 399 nts356691 AAAGGGUUCAUCCUCACCUCUCAGAAAGAGGACGAAAUCAUGAAGGUGCAGAAUAACUCUGUAAUC AUUAAUUGCGACGGUUUUUAUCUGAUUUCACUGAAGGGCUACUUUAGUCAGGAAGUUAAUAUUAGU UUGCACUACCAAAAGGACGAGGAGCCUCUCUUCCAACUAAAAAAGGUAAGAUCCGUUAAUUCCCUU AUGGUGGCCUCCUUAACUUAUAAGGACAAGGUGUAUCUGAAUGUGACCACAGAUAACACAUCCCUG GACGACUUUCAUGUAAAUGGCGGCGAGUUAAUUCUGAUACACCAGAACCCUGGCGAGUUCUGCGUG CUG OX40L Codon-optimizedAUGGUGAGCCACCGCUACCCCCGCAUCCAGAGC SEQ ID NO: 14 (TNFSF4)sequence 3 for ENSP AUCAAGGUGCAGUUCACAGAGUACAAGAAGGAG 399 nts 356691AAGGGCUUCAUCCUGACCAGCCAGAAGGAGGAU GAGAUCAUGAAGGUGCAGAACAACAGCGUCAUCAUCAACUGUGAUGGCUUCUACCUGAUCAGCCUG AAGGGCUACUUCAGCCAGGAGGUGAACAUCAGCCUGCACUACCAGAAGGAUGAGGAGCCCCUCUUC CAGCUGAAGAAGGUGCGCUCUGUGAACAGCCUGAUGGUGGCCAGCCUGACCUACAAGGACAAGGUG UACCUGAAUGUGACCACAGACAACACCAGCCUGGAUGACUUCCACGUGAAUGGAGGAGAGCUGAUC CUGAUCCACCAGAACCCUGGAGAGUUCUGUGUG CUGOX40L Codon-optimized AUGGUGAGCCACCGGUACCCCCGGAUCCAGAGC SEQ ID NO: 15(TNFSF4) sequence 4 for ENSP AUCAAGGUGCAGUUCACCGAAUACAAGAAGGAG 399 nts356691 AAGGGUUUUAUCCUGACGAGCCAGAAGGAAGACGAGAUUAUGAAGGUCCAAAACAACUCAGUCAUC AUAAACUGCGAUGGAUUUUACCUGAUCUCUCUGAAAGGGUACUUCUCCCAGGAAGUGAAUAUUAGC UUGCACUAUCAAAAAGAUGAGGAGCCUCUAUUCCAGCUCAAGAAGGUCAGAAGCGUCAAUAGUCUG AUGGUCGCAUCAUUAACCUAUAAAGACAAAGUAUAUCUAAAUGUGACGACAGACAAUACAUCCCUC GAUGAUUUUCACGUCAACGGAGGCGAACUCAUUCUGAUCCACCAGAAUCCAGGGGAAUUUUGCGUG CUG OX40L Codon-optimizedAUGGUCUCACACCGGUACCCCCGUAUCCAGAGU SEQ ID NO: 16 (TNFSF4)sequence 5 for ENSP AUUAAGGUGCAAUUCACGGAGUAUAAAAAAGAA 399 nts 356691AAGGGAUUCAUUCUGACGUCUCAGAAGGAAGAU GAGAUCAUGAAGGUCCAGAACAAUUCUGUGAUCAUUAAUUGCGAUGGAUUUUAUCUGAUUUCACUU AAAGGAUAUUUUUCCCAGGAGGUUAAUAUCAGUUUGCACUAUCAGAAAGACGAGGAGCCAUUAUUC CAGCUGAAGAAGGUGAGAUCAGUGAAUAGCCUGAUGGUUGCGUCACUGACGUAUAAAGACAAAGUU UAUCUAAACGUUACCACUGAUAAUACAUCCCUUGAUGAUUUUCAUGUGAACGGGGGUGAACUGAUC CUUAUACACCAGAACCCCGGAGAGUUCUGUGUG UUGOX40L Codon-optimized AUGGUGAGCCACAGAUACCCCAGAAUCCAGAGC SEQ ID NO: 17(TNFSF4) sequence 1 for ENSP AUCAAGGUGCAGUUCACCGAGUACAAGAAGGAG 399 nts439704 AAGGGCUUCAUCCUGACCAGCCAGAAGGAGGACGAGAUCAUGAAGGUGCAGAACAACAGCGUGAUC AUCAACUGCGACGGCUUCUACCUGAUCAGCCUGAAGGGCUACUUCAGCCAGGAGGUGAACAUCAGC CUGCACUACCAGAAGGACGAGGAGCCCCUGUUCCAGCUGAAGAAGGUGAGAAGCGUGAACAGCCUG AUGGUGGCCAGCCUGACCUACAAGGACAAGGUGUACCUGAACGUGACCACCGACAACACCAGCCUG GACGACUUCCACGUGAACGGCGGCGAGCUGAUCCUGAUCCACCAGAACCCCGGCGAGUUCUGCGUG CUG OX40L Codon-optimizedAUGGUGUCACACCGGUACCCUCGGAUCCAGUCU SEQ ID NO: 18 (TNFSF4)sequence 2 for ENSP AUUAAAGUUCAAUUUACGGAGUACAAGAAAGAA 399 nts 439704AAAGGCUUUAUCCUUACAAGCCAAAAGGAAGAC GAGAUCAUGAAAGUGCAAAACAACAGUGUGAUUAUAAAUUGUGAUGGCUUCUACCUUAUUAGUCUG AAGGGCUACUUUAGUCAGGAAGUCAAUAUUAGCCUACACUACCAGAAAGACGAGGAGCCCCUCUUU CAACUGAAAAAGGUGCGCUCCGUGAAUUCGUUGAUGGUCGCCUCUCUGACCUACAAAGAUAAGGUG UAUCUUAACGUUACUACCGACAAUACUAGUCUGGACGACUUUCACGUCAACGGAGGCGAACUUAUU CUGAUCCACCAGAACCCCGGCGAAUUCUGCGUG CUGOX40L Codon-optimized AUGGUGAGCCACCGCUACCCCCGCAUCCAGAGC SEQ ID NO: 19(TNFSF4) sequence 3 for ENSP AUCAAGGUGCAGUUCACAGAGUACAAGAAGGAG 399 nts439704 AAGGGCUUCAUCCUGACCAGCCAGAAGGAGGAUGAGAUCAUGAAGGUGCAGAACAACAGCGUCAUC AUCAACUGUGAUGGCUUCUACCUGAUCAGCCUGAAGGGCUACUUCAGCCAGGAGGUGAACAUCAGC CUGCACUACCAGAAGGAUGAGGAGCCCCUCUUCCAGCUGAAGAAGGUGCGCUCUGUGAACAGCCUG AUGGUGGCCAGCCUGACCUACAAGGACAAGGUGUACCUGAAUGUGACCACAGACAACACCAGCCUG GAUGACUUCCACGUGAAUGGAGGAGAGCUGAUCCUGAUCCACCAGAACCCUGGAGAGUUCUGUGUG CUG OX40L Codon-optimizedAUGGUGAGCCACCGGUACCCCCGGAUCCAGAGC SEQ ID NO: 20 (TNFSF4)sequence 4 for ENSP AUCAAGGUGCAGUUCACAGAGUACAAGAAGGAG 399 nts 439704AAGGGAUUUAUUCUCACAAGUCAGAAAGAAGAU GAGAUCAUGAAGGUUCAGAACAACUCAGUCAUUAUUAAUUGCGACGGAUUCUAUCUCAUUAGCCUC AAAGGCUAUUUCAGCCAGGAGGUCAAUAUCAGCCUGCACUACCAGAAGGAUGAGGAACCUCUCUUU CAGCUGAAAAAAGUCCGCUCUGUGAAUUCCCUCAUGGUCGCUUCCCUGACCUACAAGGAUAAAGUU UAUUUGAACGUUACAACAGAUAAUACAUCGCUGGACGACUUCCAUGUGAAUGGUGGCGAACUAAUU CUAAUACACCAAAAUCCAGGCGAAUUUUGUGUC CUUOX40L Codon-optimized AUGGUAUCCCAUAGAUACCCACGUAUUCAAAGC SEQ ID NO: 21(TNFSF4) sequence 5 for ENSP AUUAAGGUGCAGUUCACAGAGUACAAAAAGGAG 399 nts439704 AAGGGUUUCAUACUGACGUCACAGAAGGAGGACGAGAUAAUGAAGGUGCAGAAUAAUAGUGUGAUC AUCAAUUGUGAUGGAUUCUAUUUGAUCAGCCUCAAAGGUUAUUUCUCACAGGAAGUCAACAUUUCC CUGCACUACCAGAAGGACGAAGAGCCUUUGUUUCAGCUGAAGAAGGUGCGCUCAGUGAACAGUUUG AUGGUAGCCUCCCUAACUUAUAAAGAUAAAGUUUAUCUGAACGUGACAACCGAUAACACAUCCCUG GACGACUUUCACGUCAAUGGAGGUGAGUUAAUCCUGAUCCAUCAGAAUCCCGGAGAAUUCUGCGUU CUU

In some embodiments, the mRNA useful for the methods comprises an openreading frame encoding an extracellular domain of OX40L. In otherembodiments, the mRNA comprises an open reading frame encoding acytoplasmic domain of OX40L. In some embodiments, the mRNA comprises anopen reading frame encoding a transmembrane domain of OX40L. In certainembodiments, the mRNA comprises an open reading frame encoding anextracellular domain of OX40L and a transmembrane of OX40L. In otherembodiments, the mRNA comprises an open reading frame encoding anextracellular domain of OX40L and a cytoplasmic domain of OX40L. In yetother embodiments, the mRNA comprises an open reading frame encoding anextracellular domain of OX40L, a transmembrane of OX40L, and acytoplasmic domain of OX40L.

In some embodiments, the mRNA comprises a codon optimized sequenceencoding an OX40L polypeptide, e.g., a codon optimized sequence fromTable 1 (e.g., selected from SEQ ID NOs: 7-21).

In some embodiments, the polynucleotides comprise an mRNA encoding anOX40L polypeptide which is full length. In some embodiments, thepolynucleotides comprise an mRNA encoding a human OX40L polypeptidewhich is 183 amino acids in length. In certain embodiments, the OX40Lpolypeptide can lack at least one, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least 10, at least 14, or at least 15 amino acids atthe N-terminus or C-terminus of the OX40L polypeptide.

In some embodiments, the polynucleotide (e.g., mRNA) of the presentdisclosure is structurally modified or chemically modified. As usedherein, a “structural” modification is one in which two or more linkednucleosides are inserted, deleted, duplicated, inverted or randomized ina polynucleotide without significant chemical modification to the mRNAthemselves. Because chemical bonds will necessarily be broken andreformed to effect a structural modification, structural modificationsare of a chemical nature and hence are chemical modifications. However,structural modifications will result in a different sequence ofnucleotides. For example, the mRNA “AUCG” can be chemically modified to“AU-5meC-G”. The same mRNA can be structurally modified from “AUCG” to“AUCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in astructural modification to the polynucleotide.

In some embodiments, the polynucleotide (e.g., mRNA) of the presentdisclosure, can have a uniform chemical modification of all or any ofthe same nucleoside type or a population of modifications produced bymere downward titration of the same starting modification in all or anyof the same nucleoside type, or a measured percent of a chemicalmodification of all any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment,the polynucleotide (e.g., mRNA) encoding an OX40L polypeptide can have auniform chemical modification of two, three, or four of the samenucleoside type throughout the entire polynucleotide (e.g., mRNA) (suchas all uridines and all cytosines, etc. are modified in the same way).

When the polynucleotide (e.g., mRNA) encoding an OX40L polypeptide ofthe present disclosure are chemically and/or structurally modified themRNA can be referred to as “modified mRNA.” Non-limiting examples ofchemical modifications are described elsewhere herein.

microRNA Binding Sites

The polynucleotide (e.g., mRNA) encoding an OX40L polypeptide canfurther comprise one or more microRNA binding sites. microRNAs (ormiRNA) are 19-25 nucleotides long noncoding RNAs that bind to the 3′UTRof nucleic acid molecules and down-regulate gene expression either byreducing nucleic acid molecule stability or by inhibiting translation.

By engineering microRNA target sequences into the polynucleotides (e.g.,in a 3′UTR like region or other region) of the disclosure, one cantarget the molecule for degradation or reduced translation, provided themicroRNA in question is available. In one embodiment, the miRNA bindingsite (e.g., miR-122 binding site) binds to the corresponding maturemiRNA that is part of an active RNA-induced silencing complex (RISC)containing Dicer. In another embodiment, binding of the miRNA bindingsite to the corresponding miRNA in RISC degrades the mRNA containing themiRNA binding site or prevents the mRNA from being translated.

As used herein, the term “microRNA binding site” refers to a microRNAtarget site or a microRNA recognition site, or any nucleotide sequenceto which a microRNA binds or associates. It should be understood that“binding” can follow traditional Watson-Crick hybridization rules or canreflect any stable association of the microRNA with the target sequenceat or adjacent to the microRNA site.

Some microRNAs, e.g., miR-122, are abundant in normal tissue but arepresent in much lower levels in cancer or tumor tissue. Thus,engineering microRNA target sequences (i.e., microRNA binding site) intothe polynucleotides encoding an OX40L polypeptide (e.g., in a 3′UTR likeregion or other region) can effectively target the molecule fordegradation or reduced translation in normal tissue (where the microRNAis abundant) while providing high levels of translation in the cancer ortumor tissue (where the microRNA is present in much lower levels). Thisprovides a tumor-targeting approach for the methods and compositions ofthe disclosure.

In some embodiments, the microRNA binding site (e.g., miR-122 bindingsite) is fully complementary to miRNA (e.g., miR-122), thereby degradingthe mRNA fused to the miRNA binding site. In other embodiments, themiRNA binding site is not fully complementary to the correspondingmiRNA. In certain embodiments, the miRNA binding site (e.g., miR-122binding site) is the same length as the corresponding miRNA (e.g.,miR-122). In other embodiments, the microRNA binding site (e.g., miR-122binding site) is one nucleotide shorter than the corresponding microRNA(e.g., miR-122, which has 22 nts) at the 5′ terminus, the 3′ terminus,or both. In still other embodiments, the microRNA binding site (e.g.,miR-122 binding site) is two nucleotides shorter than the correspondingmicroRNA (e.g., miR-122) at the 5′ terminus, the 3′ terminus, or both.In yet other embodiments, the microRNA binding site (e.g., miR-122binding site) is three nucleotides shorter than the correspondingmicroRNA (e.g., miR-122) at the 5′ terminus, the 3′ terminus, or both.In some embodiments, the microRNA binding site (e.g., miR-122 bindingsite) is four nucleotides shorter than the corresponding microRNA (e.g.,miR-122) at the 5′ terminus, the 3′ terminus, or both. In otherembodiments, the microRNA binding site (e.g., miR-122 binding site) isfive nucleotides shorter than the corresponding microRNA (e.g., miR-122)at the 5′ terminus, the 3′ terminus, or both. In some embodiments, themicroRNA binding site (e.g., miR-122 binding site) is six nucleotidesshorter than the corresponding microRNA (e.g., miR-122) at the 5′terminus, the 3′ terminus, or both. In other embodiments, the microRNAbinding site (e.g., miR-122 binding site) is seven nucleotides shorterthan the corresponding microRNA (e.g., miR-122) at the 5′ terminus orthe 3′ terminus. In other embodiments, the microRNA binding site (e.g.,miR-122 binding site) is eight nucleotides shorter than thecorresponding microRNA (e.g., miR-122) at the 5′ terminus or the 3′terminus. In other embodiments, the microRNA binding site (e.g., miR-122binding site) is nine nucleotides shorter than the correspondingmicroRNA (e.g., miR-122) at the 5′ terminus or the 3′ terminus. In otherembodiments, the microRNA binding site (e.g., miR-122 binding site) isten nucleotides shorter than the corresponding microRNA (e.g., miR-122)at the 5′ terminus or the 3′ terminus. In other embodiments, themicroRNA binding site (e.g., miR-122 binding site) is eleven nucleotidesshorter than the corresponding microRNA (e.g., miR-122) at the 5′terminus or the 3′ terminus. In other embodiments, the microRNA bindingsite (e.g., miR-122 binding site) is twelve nucleotides shorter than thecorresponding microRNA (e.g., miR-122) at the 5′ terminus or the 3′terminus. The miRNA binding sites that are shorter than thecorresponding miRNAs are still capable of degrading the mRNAincorporating one or more of the miRNA binding sites or preventing themRNA from translation.

In some embodiments, the microRNA binding site (e.g., miR-122 bindingsite) has sufficient complementarity to miRNA (e.g., miR-122) so that aRISC complex comprising the miRNA (e.g., miR-122) cleaves thepolynucleotide comprising the microRNA binding site. In otherembodiments, the microRNA binding site (e.g., miR-122 binding site) hasimperfect complementarity so that a RISC complex comprising the miRNA(e.g., miR-122) induces instability in the polynucleotide comprising themicroRNA binding site. In another embodiment, the microRNA binding site(e.g., miR-122 binding site) has imperfect complementarity so that aRISC complex comprising the miRNA (e.g., miR-122) repressestranscription of the polynucleotide comprising the microRNA bindingsite. In one embodiment, the miRNA binding site (e.g., miR-122 bindingsite) has one mismatch from the corresponding miRNA (e.g., miR-122). Inanother embodiment, the miRNA binding site has two mismatches from thecorresponding miRNA. In other embodiments, the miRNA binding site hasthree mismatches from the corresponding miRNA. In other embodiments, themiRNA binding site has four mismatches from the corresponding miRNA. Insome embodiments, the miRNA binding site has five mismatches from thecorresponding miRNA. In other embodiments, the miRNA binding site hassix mismatches from the corresponding miRNA. In certain embodiments, themiRNA binding site has seven mismatches from the corresponding miRNA. Inother embodiments, the miRNA binding site has eight mismatches from thecorresponding miRNA. In other embodiments, the miRNA binding site hasnine mismatches from the corresponding miRNA. In other embodiments, themiRNA binding site has ten mismatches from the corresponding miRNA. Inother embodiments, the miRNA binding site has eleven mismatches from thecorresponding miRNA. In other embodiments, the miRNA binding site hastwelve mismatches from the corresponding miRNA.

In certain embodiments, the miRNA binding site (e.g., miR-122 bindingsite) has at least about ten contiguous nucleotides complementary to atleast about ten contiguous nucleotides of the corresponding miRNA (e.g.,miR-122), at least about eleven contiguous nucleotides complementary toat least about eleven contiguous nucleotides of the corresponding miRNA,at least about twelve contiguous nucleotides complementary to at leastabout twelve contiguous nucleotides of the corresponding miRNA, at leastabout thirteen contiguous nucleotides complementary to at least aboutthirteen contiguous nucleotides of the corresponding miRNA, or at leastabout fourteen contiguous nucleotides complementary to at least aboutfourteen contiguous nucleotides of the corresponding miRNA. In someembodiments, the miRNA binding sites have at least about fifteencontiguous nucleotides complementary to at least about fifteencontiguous nucleotides of the corresponding miRNA, at least aboutsixteen contiguous nucleotides complementary to at least about sixteencontiguous nucleotides of the corresponding miRNA, at least aboutseventeen contiguous nucleotides complementary to at least aboutseventeen contiguous nucleotides of the corresponding miRNA, at leastabout eighteen contiguous nucleotides complementary to at least abouteighteen contiguous nucleotides of the corresponding miRNA, at leastabout nineteen contiguous nucleotides complementary to at least aboutnineteen contiguous nucleotides of the corresponding miRNA, at leastabout twenty contiguous nucleotides complementary to at least abouttwenty contiguous nucleotides of the corresponding miRNA, or at leastabout twenty one contiguous nucleotides complementary to at least abouttwenty one contiguous nucleotides of the corresponding miRNA.

In some embodiments, the polynucleotides comprise an mRNA encoding anOX40L polypeptide and at least one miR122 binding site, at least twomiR122 binding sites, at least three miR122 binding sites, at least fourmiR122 binding sites, or at least five miR122 binding sites. In oneaspect, the miRNA binding site binds miR-122 or is complementary tomiR-122. In another aspect, the miRNA binding site binds to miR-122-3por miR-122-5p. In a particular aspect, the miRNA binding site comprisesa nucleotide sequence at least 80%, at least 85%, at least 90%, at least95%, or 100% identical to SEQ ID NO: 24, wherein the miRNA binding sitebinds to miR-122. In another particular aspect, the miRNA binding sitecomprises a nucleotide sequence at least 80%, at least 85%, at least90%, at least 95%, or 100% identical to SEQ ID NO: 26, wherein the miRNAbinding site binds to miR-122. These sequences are shown below in Table2.

TABLE 2 miR-122 and miR-122 binding sites SEQ ID NO. DescriptionSequence SEQ ID NO: 22 miR-122 CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUA CUGCUAGGC SEQ ID NO: 23miR-122-3p AACGCCAUUAUCACACUAAAUA SEQ ID NO: 24 miR-122-3p binding siteUAUUUAGUGUGAUAAGGCGUU SEQ ID NO: 25 miR-122-5p UGGAGUGUGACAAUGGUGUUUGSEQ ID NO: 26 miR-122-5p binding site CAAACACCAUUGUCACACUCCA

In some embodiments, a miRNA binding site (e.g., miR-122 binding site)is inserted in the polynucleotide of the disclosure in any position ofthe polynucleotide (e.g., 3′ UTR); the insertion site in thepolynucleotide can be anywhere in the polynucleotide as long as theinsertion of the miRNA binding site in the polynucleotide does notinterfere with the translation of the functional OX40L polypeptide inthe absence of the corresponding miRNA (e.g., miR122); and in thepresence of the miRNA (e.g., miR122), the insertion of the miRNA bindingsite in the polynucleotide and the binding of the miRNA binding site tothe corresponding miRNA are capable of degrading the polynucleotide orpreventing the translation of the polynucleotide. In one embodiment, amiRNA binding site is inserted in a 3′UTR of the polynucleotide.

In certain embodiments, a miRNA binding site is inserted in at leastabout 30 nucleotides downstream from the stop codon of the OX40Lencoding mRNA. In other embodiments, a miRNA binding site is inserted inat least about 10 nucleotides, at least about 15 nucleotides, at leastabout 20 nucleotides, at least about 25 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, or at least about 100nucleotides downstream from the stop codon of the polynucleotide, e.g.,the OX40L encoding mRNA. In other embodiments, a miRNA binding site isinserted in about 10 nucleotides to about 100 nucleotides, about 20nucleotides to about 90 nucleotides, about 30 nucleotides to about 80nucleotides, about 40 nucleotides to about 70 nucleotides, about 50nucleotides to about 60 nucleotides, about 45 nucleotides to about 65nucleotides downstream from the stop codon of the polynucleotide, e.g.,the OX40L encoding mRNA.

IVT Polynucleotide Architecture

In some embodiments, the polynucleotide of the present disclosurecomprising an mRNA encoding an OX40L polypeptide is an IVTpolynucleotide. Traditionally, the basic components of an mRNA moleculeinclude at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and apoly-A tail. The IVT polynucleotides of the present disclosure canfunction as mRNA but are distinguished from wild-type mRNA in theirfunctional and/or structural design features which serve, e.g., toovercome existing problems of effective polypeptide production usingnucleic-acid based therapeutics.

The primary construct of an IVT polynucleotide comprises a first regionof linked nucleotides that is flanked by a first flanking region and asecond flaking region. This first region can include, but is not limitedto, the encoded OX40L polypeptide. The first flanking region can includea sequence of linked nucleosides which function as a 5′ untranslatedregion (UTR) such as the 5′ UTR of any of the nucleic acids encoding thenative 5′ UTR of the polypeptide or a non-native 5′UTR such as, but notlimited to, a heterologous 5′ UTR or a synthetic 5′ UTR. The IVTencoding an OX40L polypeptide can comprise at its 5 terminus a signalsequence region encoding one or more signal sequences. The flankingregion can comprise a region of linked nucleotides comprising one ormore complete or incomplete 5′ UTRs sequences. The flanking region canalso comprise a 5′ terminal cap. The second flanking region can comprisea region of linked nucleotides comprising one or more complete orincomplete 3′ UTRs which can encode the native 3′ UTR of OX40L or anon-native 3′ UTR such as, but not limited to, a heterologous 3′ UTR ora synthetic 3′ UTR. The flanking region can also comprise a 3′ tailingsequence. The 3′ tailing sequence can be, but is not limited to, a polyAtail, a polyA-G quartet and/or a stem loop sequence.

Bridging the 5′ terminus of the first region and the first flankingregion is a first operational region. Traditionally, this operationalregion comprises a Start codon. The operational region can alternativelycomprise any translation initiation sequence or signal including a Startcodon.

Bridging the 3′ terminus of the first region and the second flankingregion is a second operational region. Traditionally this operationalregion comprises a Stop codon. The operational region can alternativelycomprise any translation initiation sequence or signal including a Stopcodon. Multiple serial stop codons can also be used in the IVTpolynucleotide. In some embodiments, the operation region of the presentdisclosure can comprise two stop codons. The first stop codon can be“TGA” or “UGA” and the second stop codon can be selected from the groupconsisting of “TAA,” “TGA,” “TAG,” “UAA,” “UGA” or “UAG.”

The IVT polynucleotide primary construct comprises a first region oflinked nucleotides that is flanked by a first flanking region and asecond flaking region. As used herein, the “first region” can bereferred to as a “coding region” or “region encoding” or simply the“first region.” This first region can include, but is not limited to,the encoded polypeptide of interest. In one aspect, the first region caninclude, but is not limited to, the open reading frame encoding at leastone polypeptide of interest. The open reading frame can be codonoptimized in whole or in part. The flanking region can comprise a regionof linked nucleotides comprising one or more complete or incomplete 5′UTRs sequences which can be completely codon optimized or partiallycodon optimized. The flanking region can include at least one nucleicacid sequence including, but not limited to, miR sequences, TERZAK™sequences and translation control sequences. The flanking region canalso comprise a 5′ terminal cap 138. The 5′ terminal capping region caninclude a naturally occurring cap, a synthetic cap or an optimized cap.The second flanking region can comprise a region of linked nucleotidescomprising one or more complete or incomplete 3′ UTRs. The secondflanking region can be completely codon optimized or partially codonoptimized. The flanking region can include at least one nucleic acidsequence including, but not limited to, miR sequences and translationcontrol sequences. After the second flanking region the polynucleotideprimary construct can comprise a 3′ tailing sequence. The 3′ tailingsequence can include a synthetic tailing region and/or a chainterminating nucleoside. Non-liming examples of a synthetic tailingregion include a polyA sequence, a polyC sequence, or a polyA-G quartet.Non-limiting examples of chain terminating nucleosides include 2′-Omethyl, F and locked nucleic acids (LNA).

Bridging the 5′ terminus of the first region and the first flankingregion is a first operational region. Traditionally this operationalregion comprises a Start codon. The operational region can alternativelycomprise any translation initiation sequence or signal including a Startcodon.

Bridging the 3′ terminus of the first region and the second flankingregion is a second operational region. Traditionally this operationalregion comprises a Stop codon. The operational region can alternativelycomprise any translation initiation sequence or signal including a Stopcodon. According to the present disclosure, multiple serial stop codonscan also be used.

In some embodiments, the first and second flanking regions of the IVTpolynucleotide can range independently from 15-1,000 nucleotides inlength (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500nucleotides).

In some embodiments, the tailing sequence of the IVT polynucleotide canrange from absent to 500 nucleotides in length (e.g., at least 60, 70,80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500nucleotides). Where the tailing region is a polyA tail, the length canbe determined in units of or as a function of polyA Binding Proteinbinding. In this embodiment, the polyA tail is long enough to bind atleast 4 monomers of PolyA Binding Protein. PolyA Binding Proteinmonomers bind to stretches of approximately 38 nucleotides. As such, ithas been observed that polyA tails of about 80 nucleotides and 160nucleotides are functional.

In some embodiments, the capping region of the IVT polynucleotide cancomprise a single cap or a series of nucleotides forming the cap. Inthis embodiment the capping region can be from 1 to 10, e.g. 2-9, 3-8,4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. Insome embodiments, the cap is absent.

In some embodiments, the first and second operational regions of the IVTpolynucleotide can range from 3 to 40, e.g., 5-30, 10-20, 15, or atleast 4, or 30 or fewer nucleotides in length and can comprise, inaddition to a Start and/or Stop codon, one or more signal and/orrestriction sequences.

In some embodiments, the IVT polynucleotides can be structurallymodified or chemically modified. When the IVT polynucleotides arechemically and/or structurally modified the polynucleotides can bereferred to as “modified IVT polynucleotides.”

In some embodiments, if the IVT polynucleotides are chemically modifiedthey can have a uniform chemical modification of all or any of the samenucleoside type or a population of modifications produced by meredownward titration of the same starting modification in all or any ofthe same nucleoside type, or a measured percent of a chemicalmodification of all any of the same nucleoside type but with randomincorporation, such as where all uridines are replaced by a uridineanalog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment,the IVT polynucleotides can have a uniform chemical modification of two,three, or four of the same nucleoside type throughout the entirepolynucleotide (such as all uridines and all cytosines, etc. aremodified in the same way).

In some embodiments, the IVT polynucleotides can include a sequenceencoding a self-cleaving peptide, described herein, such as but notlimited to the 2A peptide. The polynucleotide sequence of the 2A peptidein the IVT polynucleotide can be modified or codon optimized by themethods described herein and/or are known in the art. In someembodiments, this sequence can be used to separate the coding region oftwo or more polypeptides of interest in the IVT polynucleotide.

Chimeric Polynucleotide Architecture

In some embodiments, the polynucleotide of the present disclosure is achimeric polynucleotide. The chimeric polynucleotides or RNA constructsdisclosed herein maintain a modular organization similar to IVTpolynucleotides, but the chimeric polynucleotides comprise one or morestructural and/or chemical modifications or alterations which impartuseful properties to the polynucleotide. As such, the chimericpolynucleotides which are modified mRNA molecules of the presentdisclosure are termed “chimeric modified mRNA” or “chimeric mRNA.”

Chimeric polynucleotides have portions or regions which differ in sizeand/or chemical modification pattern, chemical modification position,chemical modification percent or chemical modification population andcombinations of the foregoing.

Examples of parts or regions, where the chimeric polynucleotidefunctions as an mRNA and encodes OX40L, but is not limited to,untranslated regions (UTRs, such as the 5′ UTR or 3′ UTR), codingregions, cap regions, polyA tail regions, start regions, stop regions,signal sequence regions, and combinations thereof. Regions or parts thatjoin or lie between other regions can also be designed to havesubregions.

In some embodiments, the chimeric polynucleotides of the disclosure havea structure comprising Formula I.

5′[An]x-L1-[Bo]y-L2-[Cp]z-L33′   Formula I

wherein:each of A and B independently comprises a region of linked nucleosides;either A or B or both A and B encode an OX40L polypeptide describedelsewhere herein; C is an optional region of linked nucleosides;at least one of regions A, B, or C is positionally modified, whereinsaid positionally modified region comprises at least two chemicallymodified nucleosides of one or more of the same nucleoside type ofadenosine, thymidine, guanosine, cytidine, or uridine, and wherein atleast two of the chemical modifications of nucleosides of the same typeare different chemical modifications;n, o and p are independently an integer between 15-1000;x and y are independently 1-20;z is 0-5;L1 and L2 are independently optional linker moieties, said linkermoieties being either nucleic acid based or non-nucleic acid based; andL3 is an optional conjugate or an optional linker moiety, said linkermoiety being either nucleic acid based or non-nucleic acid based.

In some embodiments, at least one of the regions of linked nucleosidesof A comprises a sequence of linked nucleosides which can function as a5′ untranslated region (UTR). The sequence of linked nucleosides can bea natural or synthetic 5′ UTR. As a non-limiting example, the chimericpolynucleotide can encode an OX40L polypeptide and the sequence oflinked nucleosides of A can encode the native 5′ UTR of the OX40Lpolypeptide or a non-heterologous 5′ UTR such as, but not limited to asynthetic UTR.

In another embodiment, at least one of the regions of linked nucleosidesof A is a cap region. The cap region can be located 5′ to a region oflinked nucleosides of A functioning as a 5′UTR. The cap region cancomprise at least one cap such as, but not limited to, Cap0, Cap1, ARCA,inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2 and Cap4.

In some embodiments, the polynucleotide of the disclosure comprises aCap1 5′UTR. In some embodiments, a polynucleotide comprising 5′UTRsequence, e.g., Cap1, for encoding an OX40L polypeptide disclosed hereinincreases expression of OX40L compared to polynucleotides encoding OX40Lcomprising a different 5′UTR (e.g., Cap0, ARCA, inosine,N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2 or Cap4). In some embodiments, a polynucleotide comprises the Cap15′UTR, wherein the polynucleotide encodes an OX40L polypeptide. In someembodiments, polynucleotide comprising the Cap1 5′UTR, increases OX40Lexpression.

In some embodiments, at least one of the regions of linked nucleosidesof B comprises at least one open reading frame of a nucleic acidsequence encoding an OX40L polypeptide. The nucleic acid sequence can becodon optimized and/or comprise at least one modification.

In some embodiments, at least one of the regions of linked nucleosidesof C comprises a sequence of linked nucleosides which can function as a3′ UTR. The sequence of linked nucleosides can be a natural or synthetic3′ UTR. As a non-limiting example, the chimeric polynucleotide canencode an OX40L polypeptide and the sequence of linked nucleosides of Ccan encode the native 3′ UTR of an OX40L polypeptide or anon-heterologous 3′ UTR such as, but not limited to a synthetic UTR.

In some embodiments, at least one of the regions of linked nucleosidesof A comprises a sequence of linked nucleosides which functions as a 5′UTR and at least one of the regions of linked nucleosides of C comprisesa sequence of linked nucleosides which functions as a 3′ UTR. In someembodiments, the 5′ UTR and the 3′ UTR can be from the same or differentspecies. In another embodiment, the 5′ UTR and the 3′ UTR can encode thenative untranslated regions from different proteins from the same ordifferent species.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure can be classified as hemimers, gapmers, wingmers, orblockmers.

As used herein, a “hemimer” is a chimeric polynucleotide comprising aregion or part which comprises half of one pattern, percent, position orpopulation of a chemical modification(s) and half of a second pattern,percent, position or population of a chemical modification(s). Chimericpolynucleotides of the present disclosure can also comprise hemimersubregions. In some embodiments, a part or region is 50% of one and 50%of another.

In some embodiments, the entire chimeric polynucleotide is 50% of oneand 50% of the other. Any region or part of any chimeric polynucleotideof the disclosure can be a hemimer. Types of hemimers include patternhemimers, population hemimers or position hemimers. By definition,hemimers are 50:50 percent hemimers.

As used herein, a “gapmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. The“gap” can comprise a region of linked nucleosides or a single nucleosidewhich differs from the chimeric nature of the two parts or regionsflanking it. The two parts or regions of a gapmer can be the same ordifferent from each other.

As used herein, a “wingmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. Unlike agapmer, the two flanking parts or regions surrounding the gap in awingmer are the same in degree or kind. Such similarity can be in thelength of number of units of different modifications or in the number ofmodifications. The wings of a wingmer can be longer or shorter than thegap. The wing parts or regions can be 20, 30, 40, 50, 60 70, 80, 90 or95% greater or shorter in length than the region which comprises thegap.

As used herein, a “blockmer” is a patterned polynucleotide where partsor regions are of equivalent size or number and type of modifications.Regions or subregions in a blockmer can be 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490 or 500, nucleosides long.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification pattern are referredto as “pattern chimeras.” Pattern chimeras can also be referred to asblockmers. Pattern chimeras are those polynucleotides having a patternof modifications within, across or among regions or parts.

Patterns of modifications within a part or region are those which startand stop within a defined region. Patterns of modifications across apart or region are those patterns which start in on part or region andend in another adjacent part or region. Patterns of modifications amongparts or regions are those which begin and end in one part or region andare repeated in a different part or region, which is not necessarilyadjacent to the first region or part.

The regions or subregions of pattern chimeras or blockmers can havesimple alternating patterns such as ABAB[AB]n where each “A” and each“B” represent different chemical modifications (at least one of thebase, sugar or backbone linker), different types of chemicalmodifications (e.g., naturally occurring and non-naturally occurring),different percentages of modifications or different populations ofmodifications. The pattern can repeat n number of times where n=3-300.Further, each A or B can represent from 1-2500 units (e.g., nucleosides)in the pattern. Patterns can also be alternating multiples such asAABBAABB[AABB]n (an alternating double multiple) orAAABBBAAABBB[AAABBB]n (an alternating triple multiple) pattern. Thepattern can repeat n number of times where n=3-300.

Different patterns can also be mixed together to form a second orderpattern. For example, a single alternating pattern can be combined witha triple alternating pattern to form a second order alternating patternA′B′. One example would be[ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB],where [ABABAB] is A′ and [AAABBBAAABBB] is B′. In like fashion, thesepatterns can be repeated n number of times, where n=3-300.

Patterns can include three or more different modifications to form anABCABC[ABC]n pattern. These three component patterns can also bemultiples, such as AABBCCAABBCC[AABBCC]n and can be designed ascombinations with other patterns such as ABCABCAABBCCABCABCAABBCC, andcan be higher order patterns.

Regions or subregions of position, percent, and population modificationsneed not reflect an equal contribution from each modification type. Theycan form series such as “1-2-3-4”, “1-2-4-8”, where each integerrepresents the number of units of a particular modification type.Alternatively, they can be odd only, such as “1-3-3-1-3-1-5” or evenonly “2-4-2-4-6-4-8” or a mixture of both odd and even number of unitssuch as “1-3-4-2-5-7-3-3-4”.

Pattern chimeras can vary in their chemical modification by degree (suchas those described above) or by kind (e.g., different modifications).

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having at least one region with two or more differentchemical modifications of two or more nucleoside members of the samenucleoside type (A, C, G, T, or U) are referred to as “positionallymodified” chimeras. Positionally modified chimeras are also referred toherein as “selective placement” chimeras or “selective placementpolynucleotides”. As the name implies, selective placement refers to thedesign of polynucleotides which, unlike polynucleotides in the art wherethe modification to any A, C, G, T or U is the same by virtue of themethod of synthesis, can have different modifications to the individualAs, Cs, Gs, Ts or Us in a polynucleotide or region thereof. For example,in a positionally modified chimeric polynucleotide, there can be two ormore different chemical modifications to any of the nucleoside types ofAs, Cs, Gs, Ts, or Us. There can also be combinations of two or more toany two or more of the same nucleoside type. For example, a positionallymodified or selective placement chimeric polynucleotide can comprise 3different modifications to the population of adenines in the moleculeand also have 3 different modifications to the population of cytosinesin the construct—all of which can have a unique, non-random, placement.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification percent are referredto as “percent chimeras.” Percent chimeras can have regions or partswhich comprise at least 1%, at least 2%, at least 5%, at least 8%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99% positional, pattern or population of modifications.Alternatively, the percent chimera can be completely modified as tomodification position, pattern, or population. The percent ofmodification of a percent chimera can be split between naturallyoccurring and non-naturally occurring modifications.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent disclosure having a chemical modification population arereferred to as “population chimeras.” A population chimera can comprisea region or part where nucleosides (their base, sugar or backbonelinkage, or combination thereof) have a select population ofmodifications. Such modifications can be selected from functionalpopulations such as modifications which induce, alter or modulate aphenotypic outcome. For example, a functional population can be apopulation or selection of chemical modifications which increase thelevel of a cytokine. Other functional populations can individually orcollectively function to decrease the level of one or more cytokines.Use of a selection of these like-function modifications in a chimericpolynucleotide would therefore constitute a “functional populationchimera.” As used herein, a “functional population chimera” can be onewhose unique functional feature is defined by the population ofmodifications as described above or the term can apply to the overallfunction of the chimeric polynucleotide itself. For example, as a wholethe chimeric polynucleotide can function in a different or superior wayas compared to an unmodified or non-chimeric polynucleotide.

It should be noted that polynucleotides which have a uniform chemicalmodification of all of any of the same nucleoside type or a populationof modifications produced by mere downward titration of the samestarting modification in all of any of the same nucleoside type, or ameasured percent of a chemical modification of all any of the samenucleoside type but with random incorporation, such as where alluridines are replaced by a uridine analog, e.g., pseudouridine or5-methoxyuridine, are not considered chimeric polynucleotides. Likewise,polynucleotides having a uniform chemical modification of two, three, orfour of the same nucleoside type throughout the entire polynucleotide(such as all uridines and all cytosines, etc. are modified in the sameway) are not considered chimeric polynucleotides. One example of apolynucleotide which is not chimeric is the canonicalpseudouridine/5-methyl cytosine modified polynucleotide. These uniformpolynucleotides are arrived at entirely via in vitro transcription (IVT)enzymatic synthesis; and due to the limitations of the synthesizingenzymes, they contain only one kind of modification at the occurrence ofeach of the same nucleoside type, i.e., adenosine (A), thymidine (T),guanosine (G), cytidine (C) or uridine (U), found in the polynucleotide.Such polynucleotides can be characterized as IVT polynucleotides.

The chimeric polynucleotides of the present disclosure can bestructurally modified or chemically modified. When the chimericpolynucleotides of the present disclosure are chemically and/orstructurally modified the polynucleotides can be referred to as“modified chimeric polynucleotides.”

The regions or parts of the chimeric polynucleotides can be separated bya linker or spacer moiety. Such linkers or spaces can be nucleic acidbased or non-nucleosidic.

In some embodiments, the chimeric polynucleotides can include a sequenceencoding a self-cleaving peptide described herein, such as, but notlimited to, a 2A peptide. The polynucleotide sequence of the 2A peptidein the chimeric polynucleotide can be modified or codon optimized by themethods described herein and/or are known in the art.

Notwithstanding the foregoing, the chimeric polynucleotides of thepresent disclosure can comprise a region or part which is notpositionally modified or not chimeric as defined herein. For example, aregion or part of a chimeric polynucleotide can be uniformly modified atone or more A, T, C, G, or U, but the polynucleotides will not beuniformly modified throughout the entire region or part.

Chimeric polynucleotides of the present disclosure can be completelypositionally modified or partially positionally modified. They can alsohave subregions which can be of any pattern or design.

In some embodiments, regions or subregions of the polynucleotides canrange from absent to 500 nucleotides in length (e.g., at least 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, or 500 nucleotides). Where the region is a polyA tail,the length can be determined in units of or as a function of polyABinding Protein binding. In this embodiment, the polyA tail is longenough to bind at least 4 monomers of PolyA Binding Protein. PolyABinding Protein monomers bind to stretches of approximately 38nucleotides. As such, it has been observed that polyA tails of about 80nucleotides to about 160 nucleotides are functional. The chimericpolynucleotides of the present disclosure which function as an mRNA neednot comprise a polyA tail.

According to the present disclosure, chimeric polynucleotides whichfunction as an mRNA can have a capping region. The capping region cancomprise a single cap or a series of nucleotides forming the cap. Inthis embodiment the capping region can be from 1 to 10, e.g. 2-9, 3-8,4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. Insome embodiments, the cap is absent.

The present disclosure contemplates chimeric polynucleotides which arecircular or cyclic. As the name implies circular polynucleotides arecircular in nature meaning that the termini are joined in some fashion,whether by ligation, covalent bond, common association with the sameprotein or other molecule or complex or by hybridization.

Chimeric polynucleotides, formulations and compositions comprisingchimeric polynucleotides, and methods of making, using and administeringchimeric polynucleotides are also described in International PatentApplication No. PCT/US2014/53907.

In some embodiments, the chimeric polynucleotide encodes an OX40Lpolypeptide. In some embodiments, the chimeric polynucleotides of thedisclosure comprise any one of the OX40L nucleic acid sequences listedin Table 1. In some embodiments the chimeric polynucleotide of thedisclosure encodes any one of the OX40L polypeptides listed in Table 1.

Circular Polynucleotide

The polynucleotide (e.g., mRNA) encoding an OX40L polypeptide can becircular or cyclic. As used herein, “circular polynucleotides” or“circP” means a single stranded circular polynucleotide which actssubstantially like, and has the properties of, an RNA. The term“circular” is also meant to encompass any secondary or tertiaryconfiguration of the circP. Circular polynucleotides are circular innature meaning that the termini are joined in some fashion, whether byligation, covalent bond, common association with the same protein orother molecule or complex or by hybridization.

Circular polynucleotides, formulations and compositions comprisingcircular polynucleotides, and methods of making, using and administeringcircular polynucleotides are also disclosed in International PatentApplication No. PCT/US2014/53904.

In some embodiments, the circular polynucleotide encodes an OX40Lpolypeptide. In some embodiments, the circular polynucleotides of thedisclosure comprise any one of the OX40L nucleic acid sequences listedin Table 1. In some embodiments, the circular polynucleotides of thedisclosure encode any one of the OX40L polypeptides listed in Table 1.In some embodiments, the circular polynucleotide increases OX40Lexpression.

Multimers of Polynucleotides

In some embodiments, multiple distinct chimeric polynucleotides and/orIVT polynucleotides can be linked together through the 3′-end usingnucleotides which are modified at the 3′-terminus. Chemical conjugationcan be used to control the stoichiometry of delivery into cells. Thiscan be controlled by chemically linking chimeric polynucleotides and/orIVT polynucleotides using a 3′-azido terminated nucleotide on onepolynucleotides species and a C5-ethynyl or alkynyl-containingnucleotide on the opposite polynucleotide species. The modifiednucleotide is added post-transcriptionally using terminal transferase(New England Biolabs, Ipswich, Mass.) according to the manufacturer'sprotocol. After the addition of the 3′-modified nucleotide, the twopolynucleotides species can be combined in an aqueous solution, in thepresence or absence of copper, to form a new covalent linkage via aclick chemistry mechanism as described in the literature.

In another example, more than two chimeric polynucleotides and/or IVTpolynucleotides can be linked together using a functionalized linkermolecule. For example, a functionalized saccharide molecule can bechemically modified to contain multiple chemical reactive groups (SH—,NH2-, N3, etc.) to react with the cognate moiety on a 3′-functionalizedmRNA molecule (i.e., a 3′-maleimide ester, 3′-NHS-ester, alkynyl). Thenumber of reactive groups on the modified saccharide can be controlledin a stoichiometric fashion to directly control the stoichiometric ratioof conjugated chimeric polynucleotides and/or IVT polynucleotides.

In some embodiments, the chimeric polynucleotides and/or IVTpolynucleotides can be linked together in a pattern. The pattern can bea simple alternating pattern such as CD[CD]x where each “C” and each “D”represent a chimeric polynucleotide, IVT polynucleotide, differentchimeric polynucleotides or different IVT polynucleotides. The patterncan repeat x number of times, where x=1-300. Patterns can also bealternating multiples such as CCDD[CCDD] x (an alternating doublemultiple) or CCCDDD[CCCDDD] x (an alternating triple multiple) pattern.The alternating double multiple or alternating triple multiple canrepeat x number of times, where x=1-300.

Conjugates and Combinations of Polynucleotides

The polynucleotide (e.g., mRNA) encoding an OX40L polypeptide can bedesigned to be conjugated to other polynucleotides, dyes, intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases, proteins, e.g., glycoproteins, orpeptides, e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell, hormones and hormonereceptors, non-peptidic species, such as lipids, lectins, carbohydrates,vitamins, cofactors, or a drug.

Conjugation can result in increased stability and/or half-life and canbe particularly useful in targeting the polynucleotides to specificsites in the cell, tissue or organism.

Polynucleotides Having Untranslated Regions (UTRs)

The polynucleotide (e.g., mRNA) encoding an OX40L polypeptide of thedisclosure can further comprise a nucleotide sequence encoding one ormore heterologous polypeptides. In one embodiment, the one or moreheterologous polypeptides improve a pharmacokinetic property orpharmacodynamics property of the OX40L polypeptide or a polynucleotideencoding the polypeptide. In another embodiment, the one or moreheterologous polypeptides comprise a polypeptide that can extend ahalf-life of the OX40L polypeptide

In one embodiment, the mRNA encodes an extracellular portion of an OX40Lpolypeptide and one or more heterologous polypeptides.

In another embodiment, the mRNA encodes an extracellular region of anOX40L polypeptide and a heterologous polypeptide. In another embodiment,the mRNA encodes a fusion protein comprising an extracellular region ofan OX40L polypeptide and a polypeptide that can extend a half-life ofthe OX40L polypeptide.

The polynucleotide (e.g., mRNA) encoding an OX40L polypeptide canfurther comprise one or more regions or parts which act or function asan untranslated region. By definition, wild type untranslated regions(UTRs) of a gene are transcribed but not translated. In mRNA, the 5′UTRstarts at the transcription start site and continues to the start codonbut does not include the start codon; whereas, the 3′UTR startsimmediately following the stop codon and continues until thetranscriptional termination signal. There is growing body of evidenceabout the regulatory roles played by the UTRs in terms of stability ofthe nucleic acid molecule and translation. The regulatory features of aUTR can be incorporated into the polynucleotides of the presentdisclosure to, among other things, enhance the stability of themolecule. The specific features can also be incorporated to ensurecontrolled down-regulation of the transcript in case they aremisdirected to undesired organs sites. Tables 3 and 4 provide a listingof exemplary UTRs which can be utilized in the polynucleotides of thepresent disclosure.

5′ UTR and Translation Initiation

In certain embodiments, the polynucleotide (e.g., mRNA) encoding anOX40L polypeptide further comprises a 5′ UTR and/or a translationinitiation sequence. Natural 5′UTRs bear features which play roles intranslation initiation. They harbor signatures like Kozak sequenceswhich are commonly known to be involved in the process by which theribosome initiates translation of many genes. 5′UTR also have been knownto form secondary structures which are involved in elongation factorbinding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides of the disclosure. Forexample, introduction of 5′ UTR of mRNA known to be upregulated incancers, such as c-myc, could be used to enhance expression of a nucleicacid molecule, such as a polynucleotides, in cancer cells. Untranslatedregions useful in the design and manufacture of polynucleotides include,but are not limited, to those disclosed in International PatentPublication No. WO 2014/164253 A2.

Shown in Table 3 is a listing of a 5′-untranslated region of thedisclosure. Variants of 5′ UTRs can be utilized wherein one or morenucleotides are added or removed to the termini, including A, U, C or G.

TABLE 3 5′-Untranslated Regions 5′UTR Name/ Identifier DescriptionSequence SEQ ID NO. 5UTR-001 Upstream UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 27 AAGAGCCACC 5UTR-002Upstream UTR GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 28AAGAGCCACC 5UTR-003 Upstream UTR GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAACSEQ ID NO: 29 GAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAAC5UTR-004 Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGSEQ ID NO: 30 CCACC 5UTR-005 Upstream UTRGGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 31 AAGAGCCACC 5UTR-006Upstream UTR GGAAUAAAAGUCUCAACACAACAUAUACAAAACAAAC SEQ ID NO: 32GAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAAC5UTR-007 Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGSEQ ID NO: 33 CCACC 5UTR-008 Upstream UTRGGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUA SEQ ID NO: 34 AAGAGCCACC 5UTR-009Upstream UTR GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 35AAGAGCCACC 5UTR-0010 Upstream UTR GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUASEQ ID NO: 36 AAGAGCCACC 5UTR-0011 Upstream UTRGGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAU SEQ ID NO: 37 AAGAGCCACC 5UTR-0012Upstream UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAU SEQ ID NO: 38AAGAGCCACC 5UTR-0013 Upstream UTR GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUSEQ ID NO: 39 AAGAGCCACC 5UTR-0014 Upstream UTRGGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAA SEQ ID NO: 40 AAGAGCCACC 5UTR-0015Upstream UTR GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 41AAGAGCCACC 5UTR-0016 Upstream UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUSEQ ID NO: 42 AAGAGCCACC 5UTR-0017 Upstream UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUU SEQ ID NO: 43 AAGAGCCACC 5UTR-0018Upstream UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAU SEQ ID NO: 44AAGAGCCACC

Other non-UTR sequences can also be used as regions or subregions withinthe polynucleotides. For example, introns or portions of intronssequences can be incorporated into regions of the polynucleotides.Incorporation of intronic sequences can increase protein production aswell as polynucleotide levels.

Combinations of features can be included in flanking regions and can becontained within other features. For example, the ORF can be flanked bya 5′ UTR which can contain a strong Kozak translational initiationsignal and/or a 3′ UTR which can include an oligo(dT) sequence fortemplated addition of a poly-A tail. 5′UTR can comprise a firstpolynucleotide fragment and a second polynucleotide fragment from thesame and/or different genes such as the 5′UTRs described in US PatentApplication Publication No. 20100293625.

These UTRs or portions thereof can be placed in the same orientation asin the transcript from which they were selected or can be altered inorientation or location. Hence a 5′ or 3′ UTR can be inverted,shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.

In some embodiments, the UTR sequences can be changed in some way inrelation to a reference sequence. For example, a 3′ or 5′ UTR can bealtered relative to a wild type or native UTR by the change inorientation or location as taught above or can be altered by theinclusion of additional nucleotides, deletion of nucleotides, swappingor transposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5′ or3′ UTR can be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR can be used asdescribed in US Patent publication 20100129877.

In some embodiments, flanking regions can be heterologous. In someembodiments, the 5′ untranslated region can be derived from a differentspecies than the 3′ untranslated region. The untranslated region canalso include translation enhancer elements (TEE). As a non-limitingexample, the TEE can include those described in US Application No.20090226470.

3′ UTR and the AU Rich Elements

In certain embodiments, the polynucleotide (e.g., mRNA) encoding anOX40L polypeptide further comprises a 3′ UTR. 3′-UTR is the section ofmRNA that immediately follows the translation termination codon andoften contains regulatory regions that post-transcriptionally influencegene expression. Regulatory regions within the 3′-UTR can influencepolyadenylation, translation efficiency, localization, and stability ofthe mRNA. In one embodiment, the 3′-UTR useful for the disclosurecomprises a binding site for regulatory proteins or microRNAs. In someembodiments, the 3′-UTR has a silencer region, which binds to repressorproteins and inhibits the expression of the mRNA. In other embodiments,the 3′-UTR comprises an AU-rich element. Proteins bind AREs to affectthe stability or decay rate of transcripts in a localized manner oraffect translation initiation. In other embodiments, the 3′-UTRcomprises the sequence AAUAAA that directs addition of several hundredadenine residues called the poly(A) tail to the end of the mRNAtranscript.

Table 4 shows a listing of 3′-untranslated regions useful for the mRNAsencoding an OX40L polypeptide. Variants of 3′ UTRs can be utilizedwherein one or more nucleotides are added or removed to the termini,including A, U, C or G.

TABLE 4A Exemplary 3′-Untranslated Regions 3′ UTR Name/ IdentifierDescription Sequence SEQ ID NO. 3′UTR-001 CreatineGCGCCUGCCCACCUGCCACCGACUGCUGGAACCCAGCCA SEQ ID NO: 45 KinaseGUGGGAGGGCCUGGCCCACCAGAGUCCUGCUCCCUCACUCCUCGCCCCGCCCCCUGUCCCAGAGUCCCACCUGGGGGCUCUCUCCACCCUUCUCAGAGUUCCAGUUUCAACCAGAGUUCCAACCAAUGGGCUCCAUCCUCUGGAUUCUGGCCAAUGAAAUAUCUCCCUGGCAGGGUCCUCUUCUUUUCCCAGAGCUCCACCCCAACCAGGAGCUCUAGUUAAUGGAGAGCUCCCAGCACACUCGGAGCUUGUGCUUUGUCUCCACGCAAAGCGAUAAAUAAAAGCAUUGGUGGCCUUUGGUCUUUGAAUAAA GCCUGAGUAGGAAGUCUAGA 3UTR-002Myoglobin GCCCCUGCCGCUCCCACCCCCACCCAUCUGGGCCCCGGG SEQ ID NO: 46UUCAAGAGAGAGCGGGGUCUGAUCUCGUGUAGCCAUAUAGAGUUUGCUUCUGAGUGUCUGCUUUGUUUAGUAGAGGUGGGCAGGAGGAGCUGAGGGGCUGGGGCUGGGGUGUUGAAGUUGGCUUUGCAUGCCCAGCGAUGCGCCUCCCUGUGGGAUGUCAUCACCCUGGGAACCGGGAGUGGCCCUUGGCUCACUGUGUUCUGCAUGGUUUGGAUCUGAAUUAAUUGUCCUUUCUUCUAAAUCCCAACCGAACUUCUUCCAACCUCCAAACUGGCUGUAACCCCAAAUCCAAGCCAUUAACUACACCUGACAGUAGCAAUUGUCUGAUUAAUCACUGGCCCCUUGAAGACAGCAGAAUGUCCCUUUGCAAUGAGGAGGAGAUCUGGGCUGGGCGGGCCAGCUGGGGAAGCAUUUGACUAUCUGGAACUUGUGUGUGCCUCCUCAGGUAUGGCAGUGACUCACCUGGUUUUAAUAAAACAACCUGCAACAUCUCAUGGUCUUUGAAUA AAGCCUGAGUAGGAAGUCUAGA 3UTR-003α-actin ACACACUCCACCUCCAGCACGCGACUUCUCAGGACGACG SEQ ID NO: 47AAUCUUCUCAAUGGGGGGGCGGCUGAGCUCCAGCCACCCCGCAGUCACUUUCUUUGUAACAACUUCCGUUGCUGCCAUCGUAAACUGACACAGUGUUUAUAACGUGUACAUACAUUAACUUAUUACCUCAUUUUGUUAUUUUUCGAAACAAAGCCCUGUGGAAGAAAAUGGAAAACUUGAAGAAGCAUUAAAGUCAUUCUGUUAAGCUGCGUAAAUGGUCUUUGAAUAAAGCCU GAGUAGGAAGUCUAGA 3UTR-004Albumin CAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAA SEQ ID NO: 48GAGAAAGAAAAUGAAGAUCAAAAGCUUAUUCAUCUGUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCAUUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAAAUGGAAAGAAUCUAAUAGAGUGGUACAGCACUGUUAUUUUUCAAAGAUGUGUUGCUAUCCUGAAAAUUCUGUAGGUUCUGUGGAAGUUCCAGUGUUCUCUCUUAUUCCACUUCGGUAGAGGAUUUCUAGUUUCUUGUGGGCUAAUUAAAUAAAUCAUUAAUACUCUUCUAAUGGUCUU UGAAUAAAGCCUGAGUAGGAAGUCUAGA3UTR-005 α-globin GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUC SEQ ID NO: 49UUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAG A 3UTR-006 G-CSFGCCAAGCCCUCCCCAUCCCAUGUAUUUAUCUCUAUUUAA SEQ ID NO: 50UAUUUAUGUCUAUUUAAGCCUCAUAUUUAAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCUCUGUGUCCUUCCCUGCAUUUCUGAGUUUCAUUCUCCUGCCUGUAGCAGUGAGAAAAAGCUCCUGUCCUCCCAUCCCCUGGACUGGGAGGUAGAUAGGUAAAUACCAAGUAUUUAUUACUAUGACUGCUCCCCAGCCCUGGCUCUGCAAUGGGCACUGGGAUGAGCCGCUGUGAGCCCCUGGUCCUGAGGGUCCCCACCUGGGACCCUUGAGAGUAUCAGGUCUCCCACGUGGGAGACAAGAAAUCCCUGUUUAAUAUUUAAACAGCAGUGUUCCCCAUCUGGGUCCUUGCACCCCUCACUCUGGCCUCAGCCGACUGCACAGCGGCCCCUGCAUCCCCUUGGCUGUGAGGCCCCUGGACAAGCAGAGGUGGCCAGAGCUGGGAGGCAUGGCCCUGGGGUCCCACGAAUUUGCUGGGGAAUCUCGUUUUUCUUCUUAAGACUUUUGGGACAUGGUUUGACUCCCGAACAUCACCGACGCGUCUCCUGUUUUUCUGGGUGGCCUCGGGACACCUGCCCUGCCCCCACGAGGGUCAGGACUGUGACUCUUUUUAGGGCCAGGCAGGUGCCUGGACAUUUGCCUUGCUGGACGGGGACUGGGGAUGUGGGAGGGAGCAGACAGGAGGAAUCAUGUCAGGCCUGUGUGUGAAAGGAAGCUCCACUGUCACCCUCCACCUCUUCACCCCCCACUCACCAGUGUCCCCUCCACUGUCACAUUGUAACUGAACUUCAGGAUAAUAAAGUGUUUGCCUCCAUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGC AUGCAUCUAGA 3UTR-007 Col1a2;ACUCAAUCUAAAUUAAAAAAGAAAGAAAUUUGAAAAAAC SEQ ID NO: 51 collagen,UUUCUCUUUGCCAUUUCUUCUUCUUCUUUUUUAACUGAA type I,AGCUGAAUCCUUCCAUUUCUUCUGCACAUCUACUUGCUU alpha 2AAAUUGUGGGCAAAAGAGAAAAAGAAGGAUUGAUCAGAGCAUUGUGCAAUACAGUUUCAUUAACUCCUUCCCCCGCUCCCCCAAAAAUUUGAAUUUUUUUUUCAACACUCUUACACCUGUUAUGGAAAAUGUCAACCUUUGUAAGAAAACCAAAAUAAAAAUUGAAAAAUAAAAACCAUAAACAUUUGCACCACUUGUGGCUUUUGAAUAUCUUCCACAGAGGGAAGUUUAAAACCCAAACUUCCAAAGGUUUAAACUACCUCAAAACACUUUCCCAUGAGUGUGAUCCACAUUGUUAGGUGCUGACCUAGACAGAGAUGAACUGAGGUCCUUGUUUUGUUUUGUUCAUAAUACAAAGGUGCUAAUUAAUAGUAUUUCAGAUACUUGAAGAAUGUUGAUGGUGCUAGAAGAAUUUGAGAAGAAAUACUCCUGUAUUGAGUUGUAUCGUGUGGUGUAUUUUUUAAAAAAUUUGAUUUAGCAUUCAUAUUUUCCAUCUUAUUCCCAAUUAAAAGUAUGCAGAUUAUUUGCCCAAAUCUUCUUCAGAUUCAGCAUUUGUUCUUUGCCAGUCUCAUUUUCAUCUUCUUCCAUGGUUCCACAGAAGCUUUGUUUCUUGGGCAAGCAGAAAAAUUAAAUUGUACCUAUUUUGUAUAUGUGAGAUGUUUAAAUAAAUUGUGAAAAAAAUGAAAUAAAGCAUGUUUGGUU UUCCAAAAGAACAUAU 3UTR-008Col6a2; CGCCGCCGCCCGGGCCCCGCAGUCGAGGGUCGUGAGCCC SEQ ID NO: 52 collagen,ACCCCGUCCAUGGUGCUAAGCGGGCCCGGGUCCCACACG type VI,GCCAGCACCGCUGCUCACUCGGACGACGCCCUGGGCCUG alpha 2CACCUCUCCAGCUCCUCCCACGGGGUCCCCGUAGCCCCGGCCCCCGCCCAGCCCCAGGUCUCCCCAGGCCCUCCGCAGGCUGCCCGGCCUCCCUCCCCCUGCAGCCAUCCCAAGGCUCCUGACCUACCUGGCCCCUGAGCUCUGGAGCAAGCCCUG ACCCAAUAAAGGCUUUGAACCCAU3UTR-009 RPNI; GGGGCUAGAGCCCUCUCCGCACAGCGUGGAGACGGGGCA SEQ ID NO: 53ribophorinI AGGAGGGGGGUUAUUAGGAUUGGUGGUUUUGUUUUGCUUUGUUUAAAGCCGUGGGAAAAUGGCACAACUUUACCUCUGUGGGAGAUGCAACACUGAGAGCCAAGGGGUGGGAGUUGGGAUAAUUUUUAUAUAAAAGAAGUUUUUCCACUUUGAAUUGCUAAAAGUGGCAUUUUUCCUAUGUGCAGUCACUCCUCUCAUUUCUAAAAUAGGGACGUGGCCAGGCACGGUGGCUCAUGCCUGUAAUCCCAGCACUUUGGGAGGCCGAGGCAGGCGGCUCACGAGGUCAGGAGAUCGAGACUAUCCUGGCUAACACGGUAAAACCCUGUCUCUACUAAAAGUACAAAAAAUUAGCUGGGCGUGGUGGUGGGCACCUGUAGUCCCAGCUACUCGGGAGGCUGAGGCAGGAGAAAGGCAUGAAUCCAAGAGGCAGAGCUUGCAGUGAGCUGAGAUCACGCCAUUGCACUCCAGCCUGGGCAACAGUGUUAAGACUCUGUCUCAAAUAUAAAUAAAUAAAUAAAUAAAUAAAUAAAUAAAUAAAAAUAAAGC GAGAUGUUGCCCUCAAA 3UTR-010LRP1; low GGCCCUGCCCCGUCGGACUGCCCCCAGAAAGCCUCCUGC SEQ ID NO: 54 densityCCCCUGCCAGUGAAGUCCUUCAGUGAGCCCCUCCCCAGC lipoproteinCAGCCCUUCCCUGGCCCCGCCGGAUGUAUAAAUGUAAAA receptor-AUGAAGGAAUUACAUUUUAUAUGUGAGCGAGCAAGCCGG relatedCAAGCGAGCACAGUAUUAUUUCUCCAUCCCCUCCCUGCC protein 1UGCUCCUUGGCACCCCCAUGCUGCCUUCAGGGAGACAGGCAGGGAGGGCUUGGGGCUGCACCUCCUACCCUCCCACCAGAACGCACCCCACUGGGAGAGCUGGUGGUGCAGCCUUCCCCUCCCUGUAUAAGACACUUUGCCAAGGCUCUCCCCUCUCGCCCCAUCCCUGCUUGCCCGCUCCCACAGCUUCCUGAGGGCUAAUUCUGGGAAGGGAGAGUUCUUUGCUGCCCCUGUCUGGAAGACGUGGCUCUGGGUGAGGUAGGCGGGAAAGGAUGGAGUGUUUUAGUUCUUGGGGGAGGCCACCCCAAACCCCAGCCCCAACUCCAGGGGCACCUAUGAGAUGGCCAUGCUCAACCCCCCUCCCAGACAGGCCCUCCCUGUCUCCAGGGCCCCCACCGAGGUUCCCAGGGCUGGAGACUUCCUCUGGUAAACAUUCCUCCAGCCUCCCCUCCCCUGGGGACGCCAAGGAGGUGGGCCACACCCAGGAAGGGAAAGCGGGCAGCCCCGUUUUGGGGACGUGAACGUUUUAAUAAUUUUUGCUGAAUUCCUUUACAACUAAAUAACACAGAUAUUGUUAUAAAUAAA AUUGU 3UTR-011 Nnt1;AUAUUAAGGAUCAAGCUGUUAGCUAAUAAUGCCACCUCU SEQ ID NO: 55 cardiotrophin-GCAGUUUUGGGAACAGGCAAAUAAAGUAUCAGUAUACAU like cytokineGGUGAUGUACAUCUGUAGCAAAGCUCUUGGAGAAAAUGA factor 1AGACUGAAGAAAGCAAAGCAAAAACUGUAUAGAGAGAUUUUUCAAAAGCAGUAAUCCCUCAAUUUUAAAAAAGGAUUGAAAAUUCUAAAUGUCUUUCUGUGCAUAUUUUUUGUGUUAGGAAUCAAAAGUAUUUUAUAAAAGGAGAAAGAACAGCCUCAUUUUAGAUGUAGUCCUGUUGGAUUUUUUAUGCCUCCUCAGUAACCAGAAAUGUUUUAAAAAACUAAGUGUUUAGGAUUUCAAGACAACAUUAUACAUGGCUCUGAAAUAUCUGACACAAUGUAAACAUUGCAGGCACCUGCAUUUUAUGUUUUUUUUUUCAACAAAUGUGACUAAUUUGAAACUUUUAUGAACUUCUGAGCUGUCCCCUUGCAAUUCAACCGCAGUUUGAAUUAAUCAUAUCAAAUCAGUUUUAAUUUUUUAAAUUGUACUUCAGAGUCUAUAUUUCAAGGGCACAUUUUCUCACUACUAUUUUAAUACAUUAAAGGACUAAAUAAUCUUUCAGAGAUGCUGGAAACAAAUCAUUUGCUUUAUAUGUUUCAUUAGAAUACCAAUGAAACAUACAACUUGAAAAUUAGUAAUAGUAUUUUUGAAGAUCCCAUUUCUAAUUGGAGAUCUCUUUAAUUUCGAUCAACUUAUAAUGUGUAGUACUAUAUUAAGUGCACUUGAGUGGAAUUCAACAUUUGACUAAUAAAAUGAGUUCAUCAUGUUGGCAAGUGAUGUGGCAAUUAUCUCUGGUGACAAAAGAGUAAAAUCAAAUAUUUCUGCCUGUUACAAAUAUCAAGGAAGACCUGCUACUAUGAAAUAGAUGACAUUAAUCUGUCUUCACUGUUUAUAAUACGGAUGGAUUUUUUUUCAAAUCAGUGUGUGUUUUGAGGUCUUAUGUAAUUGAUGACAUUUGAGAGAAAUGGUGGCUUUUUUUAGCUACCUCUUUGUUCAUUUAAGCACCAGUAAAGAUCAUGUCUUUUUAUAGAAGUGUAGAUUUUCUUUGUGACUUUGCUAUCGUGCCUAAAGCUCUAAAUAUAGGUGAAUGUGUGAUGAAUACUCAGAUUAUUUGUCUCUCUAUAUAAUUAGUUUGGUACUAAGUUUCUCAAAAAAUUAUUAACACAUGAAAGACAAUCUCUAAACCAGAAAAAGAAGUAGUACAAAUUUUGUUACUGUAAUGCUCGCGUUUAGUGAGUUUAAAACACACAGUAUCUUUUGGUUUUAUAAUCAGUUUCUAUUUUGCUGUGCCUGAGAUUAAGAUCUGUGUAUGUGUGUGUGUGUGUGUGUGCGUUUGUGUGUUAAAGCAGAAAAGACUUUUUUAAAAGUUUUAAGUGAUAAAUGCAAUUUGUUAAUUGAUCUUAGAUCACUAGUAAACUCAGGGCUGAAUUAUACCAUGUAUAUUCUAUUAGAAGAAAGUAAACACCAUCUUUAUUCCUGCCCUUUUUCUUCUCUCAAAGUAGUUGUAGUUAUAUCUAGAAAGAAGCAAUUUUGAUUUCUUGAAAAGGUAGUUCCUGCACUCAGUUUAAACUAAAAAUAAUCAUACUUGGAUUUUAUUUAUUUUUGUCAUAGUAAAAAUUUUAAUUUAUAUAUAUUUUUAUUUAGUAUUAUCUUAUUCUUUGCUAUUUGCCAAUCCUUUGUCAUCAAUUGUGUUAAAUGAAUUGAAAAUUCAUGCCCUGUUCAUUUUAUUUUACUUUAUUGGUUAGGAUAUUUAAAGGAUUUUUGUAUAUAUAAUUUCUUAAAUUAAUAUUCCAAAAGGUUAGUGGACUUAGAUUAUAAAUUAUGGCAAAAAUCUAAAAACAACAAAAAUGAUUUUUAUACAUUCUAUUUCAUUAUUCCUCUUUUUCCAAUAAGUCAUACAAUUGGUAGAUAUGACUUAUUUUAUUUUUGUAUUAUUCACUAUAUCUUUAUGAUAUUUAAGUAUAAAUAAUUAAAAAAAUUUAUUGUACCUUAUAGUCUGUCACCAAAAAAAAAAAAUUAUCUGUAGGUAGUGAAAUGCUAAUGUUGAUUUGUCUUUAAGGGCUUGUUAACUAUCCUUUAUUUUCUCAUUUGUCUUAAAUUAGGAGUUUGUGUUUAAAUUACUCAUCUAAGCAAAAAAUGUAUAUAAAUCCCAUUACUGGGUAUAUACCCAAAGGAUUAUAAAUCAUGCUGCUAUAAAGACACAUGCACACGUAUGUUUAUUGCAGCACUAUUCACAAUAGCAAAGACUUGGAACCAACCCAAAUGUCCAUCAAUGAUAGACUUGAUUAAGAAAAUGUGCACAUAUACACCAUGGAAUACUAUGCAGCCAUAAAAAAGGAUGAGUUCAUGUCCUUUGUAGGGACAUGGAUAAAGCUGGAAACCAUCAUUCUGAGCAAACUAUUGCAAGGACAGAAAACCAAACACUGCAUGUUCUCACUCAUAGGUGGGAAUUGAACAAUGAGAACACUUGGACACAAGGUGGGGAACACCACACACCAGGGCCUGUCAUGGGGUGGGGGGAGUGGGGAGGGAUAGCAUUAGGAGAUAUACCUAAUGUAAAUGAUGAGUUAAUGGGUGCAGCACACCAACAUGGCACAUGUAUACAUAUGUAGCAAACCUGCACGUUGUGCACAUGUACCCUAGAACUUAAAGUAUAAUUAAAAAAAAAAAGAAAACAGAAGCUAUUUAUAAAGAAGUUAUUUGCUGAAAUAAAUGUGAUCUUUCCCAUUAAAAAAAUAAAGAAAUUUUGGGGUAAAAAAACACAAUAUAUUGUAUUCUUGAAAAAUUCUAAGAGAGUGGAUGUGAAGUGUUCUCACCACAAAAGUGAUAACUAAUUGAGGUAAUGCACAUAUUAAUUAGAAAGAUUUUGUCAUUCCACAAUGUAUAUAUACUUAAAAAUAUGUUAUACACAAUAAAUACAUACAUUAAAAAAUAAGUAAAUGUA 3UTR-012 Col6a1;CCCACCCUGCACGCCGGCACCAAACCCUGUCCUCCCACC SEQ ID NO: 56 collagen,CCUCCCCACUCAUCACUAAACAGAGUAAAAUGUGAUGCG type VI,AAUUUUCCCGACCAACCUGAUUCGCUAGAUUUUUUUUAA alpha 1GGAAAAGCUUGGAAAGCCAGGACACAACGCUGCUGCCUGCUUUGUGCAGGGUCCUCCGGGGCUCAGCCCUGAGUUGGCAUCACCUGCGCAGGGCCCUCUGGGGCUCAGCCCUGAGCUAGUGUCACCUGCACAGGGCCCUCUGAGGCUCAGCCCUGAGCUGGCGUCACCUGUGCAGGGCCCUCUGGGGCUCAGCCCUGAGCUGGCCUCACCUGGGUUCCCCACCCCGGGCUCUCCUGCCCUGCCCUCCUGCCCGCCCUCCCUCCUGCCUGCGCAGCUCCUUCCCUAGGCACCUCUGUGCUGCAUCCCACCAGCCUGAGCAAGACGCCCUCUCGGGGCCUGUGCCGCACUAGCCUCCCUCUCCUCUGUCCCCAUAGCUGGUUUUUCCCACCAAUCCUCACCUAACAGUUACUUUACAAUUAAACUCAAAGCAAGCUCUUCUCCUCAGCUUGGGGCAGCCAUUGGCCUCUGUCUCGUUUUGGGAAACCAAGGUCAGGAGGCCGUUGCAGACAUAAAUCUCGGCGACUCGGCCCCGUCUCCUGAGGGUCCUGCUGGUGACCGGCCUGGACCUUGGCCCUACAGCCCUGGAGGCCGCUGCUGACCAGCACUGACCCCGACCUCAGAGAGUACUCGCAGGGGCGCUGGCUGCACUCAAGACCCUCGAGAUUAACGGUGCUAACCCCGUCUGCUCCUCCCUCCCGCAGAGACUGGGGCCUGGACUGGACAUGAGAGCCCCUUGGUGCCACAGAGGGCUGUGUCUUACUAGAAACAACGCAAACCUCUCCUUCCUCAGAAUAGUGAUGUGUUCGACGUUUUAUCAAAGGCCCCCUUUCUAUGUUCAUGUUAGUUUUGCUCCUUCUGUGUUUUUUUCUGAACCAUAUCCAUGUUGCUGACUUUUCC AAAUAAAGGUUUUCACUCCUCUC 3UTR-013Calr; AGAGGCCUGCCUCCAGGGCUGGACUGAGGCCUGAGCGCU SEQ ID NO: 57 calreticluinCCUGCCGCAGAGCUGGCCGCGCCAAAUAAUGUCUCUGUGAGACUCGAGAACUUUCAUUUUUUUCCAGGCUGGUUCGGAUUUGGGGUGGAUUUUGGUUUUGUUCCCCUCCUCCACUCUCCCCCACCCCCUCCCCGCCCUUUUUUUUUUUUUUUUUUAAACUGGUAUUUUAUCUUUGAUUCUCCUUCAGCCCUCACCCCUGGUUCUCAUCUUUCUUGAUCAACAUCUUUUCUUGCCUCUGUCCCCUUCUCUCAUCUCUUAGCUCCCCUCCAACCUGGGGGGCAGUGGUGUGGAGAAGCCACAGGCCUGAGAUUUCAUCUGCUCUCCUUCCUGGAGCCCAGAGGAGGGCAGCAGAAGGGGGUGGUGUCUCCAACCCCCCAGCACUGAGGAAGAACGGGGCUCUUCUCAUUUCACCCCUCCCUUUCUCCCCUGCCCCCAGGACUGGGCCACUUCUGGGUGGGGCAGUGGGUCCCAGAUUGGCUCACACUGAGAAUGUAAGAACUACAAACA AAAUUUCUAUUAAAUUAAAUUUUGUGUCUCC3UTR-014 Colla1; CUCCCUCCAUCCCAACCUGGCUCCCUCCCACCCAACCAA SEQ ID NO: 58collagen, CUUUCCCCCCAACCCGGAAACAGACAAGCAACCCAAACU type I,GAACCCCCUCAAAAGCCAAAAAAUGGGAGACAAUUUCAC alpha 1AUGGACUUUGGAAAAUAUUUUUUUCCUUUGCAUUCAUCUCUCAAACUUAGUUUUUAUCUUUGACCAACCGAACAUGACCAAAAACCAAAAGUGCAUUCAACCUUACCAAAAAAAAAAAAAAAAAAAGAAUAAAUAAAUAACUUUUUAAAAAAGGAAGCUUGGUCCACUUGCUUGAAGACCCAUGCGGGGGUAAGUCCCUUUCUGCCCGUUGGGCUUAUGAAACCCCAAUGCUGCCCUUUCUGCUCCUUUCUCCACACCCCCCUUGGGGCCUCCCCUCCACUCCUUCCCAAAUCUGUCUCCCCAGAAGACACAGGAAACAAUGUAUUGUCUGCCCAGCAAUCAAAGGCAAUGCUCAAACACCCAAGUGGCCCCCACCCUCAGCCCGCUCCUGCCCGCCCAGCACCCCCAGGCCCUGGGGGACCUGGGGUUCUCAGACUGCCAAAGAAGCCUUGCCAUCUGGCGCUCCCAUGGCUCUUGCAACAUCUCCCCUUCGUUUUUGAGGGGGUCAUGCCGGGGGAGCCACCAGCCCCUCACUGGGUUCGGAGGAGAGUCAGGAAGGGCCACGACAAAGCAGAAACAUCGGAUUUGGGGAACGCGUGUCAAUCCCUUGUGCCGCAGGGCUGGGCGGGAGAGACUGUUCUGUUCCUUGUGUAACUGUGUUGCUGAAAGACUACCUCGUUCUUGUCUUGAUGUGUCACCGGGGCAACUGCCUGGGGGCGGGGAUGGGGGCAGGGUGGAAGCGGCUCCCCAUUUUAUACCAAAGGUGCUACAUCUAUGUGAUGGGUGGGGUGGGGAGGGAAUCACUGGUGCUAUAGAAAUUGAGAUGCCCCCCCAGGCCAGCAAAUGUUCCUUUUUGUUCAAAGUCUAUUUUUAUUCCUUGAUAUUUUUCUUUUUUUUUUUUUUUUUUUGUGGAUGGGGACUUGUGAAUUUUUCUAAAGGUGCUAUUUAACAUGGGAGGAGAGCGUGUGCGGCUCCAGCCCAGCCCGCUGCUCACUUUCCACCCUCUCUCCACCUGCCUCUGGCUUCUCAGGCCUCUGCUCUCCGACCUCUCUCCUCUGAAACCCUCCUCCACAGCUGCAGCCCAUCCUCCCGGCUCCCUCCUAGUCUGUCCUGCGUCCUCUGUCCCCGGGUUUCAGAGACAACUUCCCAAAGCACAAAGCAGUUUUUCCCCCUAGGGGUGGGAGGAAGCAAAAGACUCUGUACCUAUUUUGUAUGUGUAUAAUAAUUUGAGAUGUUUUUAAUUAUUUUGAUUGCUGGAAUAAAGCAUGUGGAAAUGACCCAAACAUAAUCCGCAGUGGCCUCCUAAUUUCCUUCUUUGGAGUUGGGGGAGGGGUAGACAUGGGGAAGGGGCUUUGGGGUGAUGGGCUUGCCUUCCAUUCCUGCCCUUUCCCUCCCCACUAUUCUCUUCUAGAUCCCUCCAUAACCCCACUCCCCUUUCUCUCACCCUUCUUAUACCGCAAACCUUUCUACUUCCUCUUUCAUUUUCUAUUCUUGCAAUUUCCUUGCACCUUUUCCAAAUCCUCUUCUCCCCUGCAAUACCAUACAGGCAAUCCACGUGCACAACACACACACACACUCUUCACAUCUGGGGUUGUCCAAACCUCAUACCCACUCCCCUUCAAGCCCAUCCACUCUCCACCCCCUGGAUGCCCUGCACUUGGUGGCGGUGGGAUGCUCAUGGAUACUGGGAGGGUGAGGGGAGUGGAACCCGUGAGGAGGACCUGGGGGCCUCUCCUUGAACUGACAUGAAGGGUCAUCUGGCCUCUGCUCCCUUCUCACCCACGCUGACCUCCUGCCGAAGGAGCAACGCAACAGGAGAGGGGUCUGCUGAGCCUGGCGAGGGUCUGGGAGGGACCAGGAGGAAGGCGUGCUCCCUGCUCGCUGUCCUGGCCCUGGGGGAGUGAGGGAGACAGACACCUGGGAGAGCUGUGGGGAAGGCACUCGCACCGUGCUCUUGGGAAGGAAGGAGACCUGGCCCUGCUCACCACGGACUGGGUGCCUCGACCUCCUGAAUCCCCAGAACACAACCCCCCUGGGCUGGGGUGGUCUGGGGAACCAUCGUGCCCC CGCCUCCCGCCUACUCCUUUUUAAGCUU3UTR-015 Plod1; UUGGCCAGGCCUGACCCUCUUGGACCUUUCUUCUUUGCC SEQ ID NO: 59procollagen- GACAACCACUGCCCAGCAGCCUCUGGGACCUCGGGGUCC lysine,CAGGGAACCCAGUCCAGCCUCCUGGCUGUUGACUUCCCA 2-oxoglutarateUUGCUCUUGGAGCCACCAAUCAAAGAGAUUCAAAGAGAU 5-dioxygenase 1UCCUGCAGGCCAGAGGCGGAACACACCUUUAUGGCUGGGGCUCUCCGUGGUGUUCUGGACCCAGCCCCUGGAGACACCAUUCACUUUUACUGCUUUGUAGUGACUCGUGCUCUCCAACCUGUCUUCCUGAAAAACCAAGGCCCCCUUCCCCCACCUCUUCCAUGGGGUGAGACUUGAGCAGAACAGGGGCUUCCCCAAGUUGCCCAGAAAGACUGUCUGGGUGAGAAGCCAUGGCCAGAGCUUCUCCCAGGCACAGGUGUUGCACCAGGGACUUCUGCUUCAAGUUUUGGGGUAAAGACACCUGGAUCAGACUCCAAGGGCUGCCCUGAGUCUGGGACUUCUGCCUCCAUGGCUGGUCAUGAGAGCAAACCGUAGUCCCCUGGAGACAGCGACUCCAGAGAACCUCUUGGGAGACAGAAGAGGCAUCUGUGCACAGCUCGAUCUUCUACUUGCCUGUGGGGAGGGGAGUGACAGGUCCACACACCACACUGGGUCACCCUGUCCUGGAUGCCUCUGAAGAGAGGGACAGACCGUCAGAAACUGGAG AGUUUCUAUUAAAGGUCAUUUAAACCA3UTR-016 Nucb1; UCCUCCGGGACCCCAGCCCUCAGGAUUCCUGAUGCUCCA SEQ ID NO: 60nucleobindin 1 AGGCGACUGAUGGGCGCUGGAUGAAGUGGCACAGUCAGCUUCCCUGGGGGCUGGUGUCAUGUUGGGCUCCUGGGGCGGGGGCACGGCCUGGCAUUUCACGCAUUGCUGCCACCCCAGGUCCACCUGUCUCCACUUUCACAGCCUCCAAGUCUGUGGCUCUUCCCUUCUGUCCUCCGAGGGGCUUGCCUUCUCUCGUGUCCAGUGAGGUGCUCAGUGAUCGGCUUAACUUAGAGAAGCCCGCCCCCUCCCCUUCUCCGUCUGUCCCAAGAGGGUCUGCUCUGAGCCUGCGUUCCUAGGUGGCUCGGCCUCAGCUGCCUGGGUUGUGGCCGCCCUAGCAUCCUGUAUGCCCACAGCUACUGGAAUCCCCGCUGCUGCUCCGGGCCAAGCUUCUGGUUGAUUAAUGAGGGCAUGGGGUGGUCCCUCAAGACCUUCCCCUACCUUUUGUGGAACCAGUGAUGCCUCAAAGACAGUGUCCCCUCCACAGCUGGGUGCCAGGGGCAGGGGAUCCUCAGUAUAGCCGGUGAACCCUGAUACCAGGAGCCUGGGCCUCCCUGAACCCCUGGCUUCCAGCCAUCUCAUCGCCAGCCUCCUCCUGGACCUCUUGGCCCCCAGCCCCUUCCCCACACAGCCCCAGAAGGGUCCCAGAGCUGACCCCACUCCAGGACCUAGGCCCAGCCCCUCAGCCUCAUCUGGAGCCCCUGAAGACCAGUCCCACCCACCUUUCUGGCCUCAUCUGACACUGCUCCGCAUCCUGCUGUGUGUCCUGUUCCAUGUUCCGGU UCCAUCCAAAUACACUUUCUGGAACAAA3UTR-017 α-globin GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC SEQ ID NO: 61UCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCC CGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC3UTR-018 Downstream UAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUSEQ ID NO: 62 UTR UGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

In certain embodiments, the 3′ UTR sequence useful for the disclosurecomprises a nucleotide sequence at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% identical to a sequence selected from the group consistingof SEQ ID NOS: 45-62 and any combination thereof. In a particularembodiment, the 3′ UTR sequence further comprises a miRNA binding site,e.g., miR122 binding site. In other embodiments, a 3′UTR sequence usefulfor the disclosure comprises 3′ UTR-018 (SEQ ID NO: 62).

In certain embodiments, the 3′ UTR sequence comprises one or more miRNAbinding sites, e.g., miR-122 binding sites, or any other heterologousnucleotide sequences therein, without disrupting the function of the 3′UTR. Some examples of 3′ UTR sequences comprising a miRNA binding siteare listed in Table 4B.

TABLE 4B Exemplary 3′ UTR with miRNA Binding Sites 3′UTR Identifier/Name/ miRNA BS Description Sequence SEQ ID NO. 3UTR-018 + DownstreamUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC SEQ ID NO: 63 miR-122-5p UTRCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG binding CACCCGUACCCCCCAAACACCAUUGUCACACUCCA G site UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 3UTR-018 +Downstream UAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC SEQ ID NO: 64 miR-122-3pUTR CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG binding CACCCGUACCCCCUAUUUAGUGUGAUAAUGGCGUU G site UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC *miRNAbinding site is bolded and underlined.

In certain embodiments, the 3′ UTR sequence useful for the disclosurecomprises a nucleotide sequence at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% identical to the sequence set forth as SEQ ID NO: 63 or64.

Regions Having a 5′ Cap

The polynucleotide comprising an mRNA encoding an OX40L polypeptide canfurther comprise a 5′ cap. The 5′ cap useful for the OX40L encoding mRNAcan bind the mRNA Cap Binding Protein (CBP), thereby increasing mRNAstability. The cap can further assist the removal of 5′ proximal intronsremoval during mRNA splicing.

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure comprises a non-hydrolyzablecap structure preventing decapping and thus increasing mRNA half-life.Because cap structure hydrolysis requires cleavage of 5′-ppp-5′phosphorodiester linkages, modified nucleotides can be used during thecapping reaction. For example, a Vaccinia Capping Enzyme from NewEngland Biolabs (Ipswich, Mass.) can be used with α-thio-guanosinenucleotides according to the manufacturer's instructions to create aphosphorothioate linkage in the 5′-ppp-5′ cap. Additional modifiedguanosine nucleotides can be used such as α-methyl-phosphonate andseleno-phosphate nucleotides.

In certain embodiments, the 5′ cap comprises 2′-O-methylation of theribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides on the2′-hydroxyl group of the sugar ring. In other embodiments, the caps forthe OX40L encoding mRNA include cap analogs, which herein are alsoreferred to as synthetic cap analogs, chemical caps, chemical capanalogs, or structural or functional cap analogs, differ from natural(i.e. endogenous, wild-type or physiological) 5′-caps in their chemicalstructure, while retaining cap function. Cap analogs can be chemically(i.e. non-enzymatically) or enzymatically synthesized and/or linked tothe polynucleotides of the disclosure.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which can equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-0atom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped polynucleotide. The N7- and 3′-O-methlyatedguanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In some embodiments, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog can be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m^(3′-O)G(5′)ppp(5′)G cap analog (See, e.g.,the various cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574). In another embodiment, a cap analog of the presentdisclosure is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotideor a region thereof, in an in vitro transcription reaction, up to 20% oftranscripts can remain uncapped. This, as well as the structuraldifferences of a cap analog from an endogenous 5′-cap structure ofnucleic acids produced by the endogenous, cellular transcriptionmachinery, can lead to reduced translational competency and reducedcellular stability.

The OX40L encoding mRNA of the disclosure can also be cappedpost-manufacture (whether IVT or chemical synthesis), using enzymes, inorder to generate more authentic 5′-cap structures. As used herein, thephrase “more authentic” refers to a feature that closely mirrors ormimics, either structurally or functionally, an endogenous or wild typefeature. That is, a “more authentic” feature is better representative ofan endogenous, wild-type, natural or physiological cellular functionand/or structure as compared to synthetic features or analogs, etc., ofthe prior art, or which outperforms the corresponding endogenous,wild-type, natural or physiological feature in one or more respects.Non-limiting examples of more authentic 5′cap structures of the presentdisclosure are those which, among other things, have enhanced binding ofcap binding proteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of apolynucleotide and a guanine cap nucleotide wherein the cap guaninecontains an N7 methylation and the 5′-terminal nucleotide of the mRNAcontains a 2′-O-methyl. Such a structure is termed the Cap1 structure.This cap results in a higher translational-competency and cellularstability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′cap analog structures known inthe art. Cap structures include, but are not limited to,7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)N1mpNp (cap 1), and7mG(5′)-ppp(5′)N1mpN2mp (cap 2).

According to the present disclosure, 5′ terminal caps can includeendogenous caps or cap analogs. According to the present disclosure, a5′ terminal cap can comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Poly-A Tails

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide further comprises a poly A tail. In furtherembodiments, terminal groups on the poly-A tail can be incorporated forstabilization. In other embodiments, a poly-A tail comprises des-3′hydroxyl tails. The useful poly-A tails can also include structuralmoieties or 2′-Omethyl modifications as taught by Junjie Li, et al.(Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005).

In one embodiment, the length of a poly-A tail, when present, is greaterthan 30 nucleotides in length. In another embodiment, the poly-A tail isgreater than 35 nucleotides in length (e.g., at least or greater thanabout 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200,1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000nucleotides). In some embodiments, the polynucleotide or region thereofincludes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50,from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000,from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from2,000 to 2,500, and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the lengthof the overall polynucleotide or the length of a particular region ofthe polynucleotide. This design can be based on the length of a codingregion, the length of a particular feature or region or based on thelength of the ultimate product expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotide or featurethereof. The poly-A tail can also be designed as a fraction of thepolynucleotides to which it belongs. In this context, the poly-A tailcan be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct, a construct region or the total length of theconstruct minus the poly-A tail. Further, engineered binding sites andconjugation of polynucleotides for Poly-A binding protein can enhanceexpression.

Additionally, multiple distinct polynucleotides can be linked togethervia the PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

In some embodiments, the polynucleotides of the present disclosure aredesigned to include a polyA-G Quartet region. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantpolynucleotide is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein production froman mRNA equivalent to at least 75% of that seen using a poly-A tail of120 nucleotides alone.

Start Codon Region

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure further comprises regionsthat are analogous to or function like a start codon region.

In some embodiments, the translation of a polynucleotide initiates on acodon which is not the start codon AUG. Translation of thepolynucleotide can initiate on an alternative start codon such as, butnot limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU,TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 andMatsuda and Mauro PLoS ONE, 2010 5:11). As a non-limiting example, thetranslation of a polynucleotide begins on the alternative start codonACG. As another non-limiting example, polynucleotide translation beginson the alternative start codon CTG or CUG. As yet another non-limitingexample, the translation of a polynucleotide begins on the alternativestart codon GTG or GUG.

Nucleotides flanking a codon that initiates translation such as, but notlimited to, a start codon or an alternative start codon, are known toaffect the translation efficiency, the length and/or the structure ofthe polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5:11).Masking any of the nucleotides flanking a codon that initiatestranslation can be used to alter the position of translation initiation,translation efficiency, length and/or structure of a polynucleotide.

In some embodiments, a masking agent is used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) polynucleotides and exon-junctioncomplexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agentsLNA polynucleotides and EJCs (PLoS ONE, 2010 5:11)).

In another embodiment, a masking agent is used to mask a start codon ofa polynucleotide in order to increase the likelihood that translationwill initiate on an alternative start codon. In some embodiments, amasking agent is used to mask a first start codon or alternative startcodon in order to increase the chance that translation will initiate ona start codon or alternative start codon downstream to the masked startcodon or alternative start codon.

In some embodiments, a start codon or alternative start codon is locatedwithin a perfect complement for a miR binding site. The perfectcomplement of a miR binding site can help control the translation,length and/or structure of the polynucleotide similar to a maskingagent. As a non-limiting example, the start codon or alternative startcodon is located in the middle of a perfect complement for a miR-122binding site. The start codon or alternative start codon can be locatedafter the first nucleotide, second nucleotide, third nucleotide, fourthnucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide,eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventhnucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenthnucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenthnucleotide, eighteenth nucleotide, nineteenth nucleotide, twentiethnucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide is removedfrom the polynucleotide sequence in order to have the translation of thepolynucleotide begin on a codon which is not the start codon.Translation of the polynucleotide can begin on the codon following theremoved start codon or on a downstream start codon or an alternativestart codon. In a non-limiting example, the start codon ATG or AUG isremoved as the first 3 nucleotides of the polynucleotide sequence inorder to have translation initiate on a downstream start codon oralternative start codon. The polynucleotide sequence where the startcodon was removed can further comprise at least one masking agent forthe downstream start codon and/or alternative start codons in order tocontrol or attempt to control the initiation of translation, the lengthof the polynucleotide and/or the structure of the polynucleotide.

Stop Codon Region

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure can further comprise atleast one stop codon or at least two stop codons before the 3′untranslated region (UTR). The stop codon can be selected from UGA, UAA,and UAG. In some embodiments, the polynucleotides of the presentdisclosure include the stop codon UGA and one additional stop codon. Ina further embodiment the addition stop codon can be UAA. In anotherembodiment, the polynucleotides of the present disclosure include threestop codons, four stop codons, or more.

Polynucleotide Comprising mRNA Encoding an OX40L Polypeptide

In certain embodiments, the polynucleotide comprising an mRNA encodingan OX40L polypeptide of the present disclosure comprises (i) an mRNAencoding an OX40L polypeptide, such as the sequences provided in Table 1above, (ii) a miR-122 binding site, such as the sequences provided inTable 2 above, (iii) a 5′ UTR, such as the sequences provided in Table 3above, and (iv) a 3′ UTR, such as the sequences provided in Table 4A or4B above. In a particular embodiment, the polynucleotide of the presentdisclosure comprises a sequence set forth in Table 5A below (SEQ ID NO:65).

TABLE 5A Polynucleotides comprising an mRNA encoding anOX40L polypeptide and a miR-122 binding site SEQ ID NO. DescriptionSequence SEQ ID NO: 65 mRNA sequence:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC Human OX40L withACCAUGGAAAGGGUCCAACCCCUGGAAGAGAAUGUGGGAAAUGC 5′-UTR, 3′-UTR, andAGCCAGGCCAAGAUUCGAGAGGAACAAGCUAUUGCUGGUGGCCU miR-122 binding siteCUGUAAUUCAGGGACUGGGGCUGCUCCUGUGCUUCACCUACAUCUGCCUGCACUUCUCUGCUCUUCAGGUAUCACAUCGGUAUCCUCGAAUUCAAAGUAUCAAAGUACAAUUUACCGAAUAUAAGAAGGAGAAAGGUUUCAUCCUCACUUCCCAAAAGGAGGAUGAAAUCAUGAAGGUGCAGAACAACUCAGUCAUCAUCAACUGUGAUGGGUUUUAUCUCAUCUCCCUGAAGGGCUACUUCUCCCAGGAAGUCAACAUUAGCCUUCAUUACCAGAAGGAUGAGGAGCCCCUCUUCCAACUGAAGAAGGUCAGGUCUGUCAACUCCUUGAUGGUGGCCUCUCUGACUUACAAAGACAAAGUCUACUUGAAUGUGACCACUGACAAUACCUCCCUGGAUGACUUCCAUGUGAAUGGCGGAGAACUGAUUCUUAUCCAUCAAAAUCCUGGUGAAUUCUGUGUCCUUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Additional polynucleotides comprising an mRNA, a miR-122 binding site, a5′ UTR, and a 3′ UTR are shown below in Table 5B.

TABLE 5BAdditional polynucleotides comprising an mRNA and a miR-122 binding siteSEQ ID NO. Description Sequence SEQ ID NO: 66 mRNA sequence:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC murine OX40L withACCAUGGAAGGGGAAGGGGUUCAACCCCUGGAUGAGAAUCUGGA 5′-UTR, 3′-UTR, andAAACGGAUCAAGGCCAAGAUUCAAGUGGAAGAAGACGCUAAGGC miR-122 binding siteUGGUGGUCUCUGGGAUCAAGGGAGCAGGGAUGCUUCUGUGCUUCAUCUAUGUCUGCCUGCAACUCUCUUCCUCUCCGGCAAAGGACCCUCCAAUCCAAAGACUCAGAGGAGCAGUUACCAGAUGUGAGGAUGGGCAACUAUUCAUCAGCUCAUACAAGAAUGAGUAUCAAACUAUGGAGGUGCAGAACAAUUCGGUUGUCAUCAAGUGCGAUGGGCUUUAUAUCAUCUACCUGAAGGGCUCCUUUUUCCAGGAGGUCAAGAUUGACCUUCAUUUCCGGGAGGAUCAUAAUCCCAUCUCUAUUCCAAUGCUGAACGAUGGUCGAAGGAUUGUCUUCACUGUGGUGGCCUCUUUGGCUUUCAAAGAUAAAGUUUACCUGACUGUAAAUGCUCCUGAUACUCUCUGCGAACACCUCCAGAUAAAUGAUGGGGAGCUGAUUGUUGUCCAGCUAACGCCUGGAUACUGUGCUCCUGAAGGAUCUUACCACAGCACUGUGAACCAAGUACCACUGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC SEQ ID 67 mRNA sequence: non-GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACAG translatable FIX withCGCGUCAACAUUGCCGAAUCGCCGGGACUCAUCACAAUCUGCCU 5′-UTR, 3′UTR andCUUGGGUUAUCUCUUGUCGGCAGAUACCUUCUUGGAUCACGAAA miR-122 binding siteACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGG (NST FIX)AAACUUGAGGAGUUUCAGGGCAAUCUUGAACGAGACGAGGAGAACUCCUUUGAGGAGGCGAGGGAAUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGGGGACCAGUCGAAUCCCCUCAGGGGAUCUAAAGACAUCAAUAGCUACUGCCCGUUUGGGUUUGAAGGGAAGAACUAGCUGACCAACAUCAAAAACGGACGCUAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUCUCCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCCGAGCCCGCAGUCCCGUUCCCUUGGAGGAGCUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAACUUUACGAGGGUCGUAGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUAUUGAACGGAAAAGUCGCCUUUUGAGGUUCCAUUGUCAACGAGAAGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAAAAAUCACGGUAGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAUUCCAUAAGGGUAGAUCCGCACUGUUGCAAUACCUCAGGCCCCUCGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUUUCUCGGGAUUCCAAGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCGGGAGAGGCGAAGGGGAACAUCUACACUAAAUCACGGUUCAAUUGGAUCAAGGAAAAGACGAAACUCACGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC SEQ ID NO: 68mRNA sequence: non- GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAACCtranslatable OX40L with CGCAAGGAAGGGGAAGGGGUUCAACCCCUGGAAGAGAAUCUGGA5′-UTR, 3′UTR, and AAACGGAUCAAGGCCAAGAUUCAAGAGGAAGAAGACGCUAAGGCmiR-122 binding site UGGAGGUCUCUGGGAUCAAGGGAGCAGGGAAGCUUCUGAGCUUC(NST OX40L) AUCUAAGUCUGCCUGCAACUCUCUUCCUCUCCGGCAAAGGACCCUCCAAUCCAAAGACUCAGAGGAGCAGUUACCAGAAGAGAGGAAGGGCAACUAUUCAUCAGCUCAUACAAGAAAGAGUAUCAAACUAAGGAGGAGCAGAACAAUUCGGUUGUCAUCAAGAGCGAAGGGCUUUAUAUCAUCUACCUGAAGGGCUCCUUUUUCCAGGAGGUCAAGAUUGACCUUCAUUUCCGGGAGGAUCAUAAUCCCAUCUCUAUUCCAAAGCUGAACGAAGGUCGAAGGAUUGUCUUCACUGAGGAGGCCUCUUUGGCUUUCAAAGAUAAAGUUUACCUGACUGUAAAAGCUCCUGAUACUCUCUGCGAACACCUCCAGAUAAAAGAAGGGGAGCUGAUUGUUGUCCAGCUAACGCCUGGAUACUGAGCUCCUGAAGGAUCUUACCACAGCACUGAGAACCAAGUACCACUGUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC SEQ ID 69 mRNA sequence:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC Firefly luciferase withACCAUGGAAGAUGCGAAGAACAUCAAGAAGGGACCUGCCCCGUU 5′-UTR, 3′-UTR, andUUACCCUUUGGAGGACGGUACAGCAGGAGAACAGCUCCACAAGG miR-122 binding siteCGAUGAAACGCUACGCCCUGGUCCCCGGAACGAUUGCGUUUACCGAUGCACAUAUUGAGGUAGACAUCACAUACGCAGAAUACUUCGAAAUGUCGGUGAGGCUGGCGGAAGCGAUGAAGAGAUAUGGUCUUAACACUAAUCACCGCAUCGUGGUGUGUUCGGAGAACUCAUUGCAGUUUUUCAUGCCGGUCCUUGGAGCACUUUUCAUCGGGGUCGCAGUCGCGCCAGCGAACGACAUCUACAAUGAGCGGGAACUCUUGAAUAGCAUGGGAAUCUCCCAGCCGACGGUCGUGUUUGUCUCCAAAAAGGGGCUGCAGAAAAUCCUCAACGUGCAGAAGAAGCUCCCCAUUAUUCAAAAGAUCAUCAUUAUGGAUAGCAAGACAGAUUACCAAGGGUUCCAGUCGAUGUAUACCUUUGUGACAUCGCAUUUGCCGCCAGGGUUUAACGAGUAUGACUUCGUCCCCGAGUCAUUUGACAGAGAUAAAACCAUCGCGCUGAUUAUGAAUUCCUCGGGUAGCACCGGUUUGCCAAAGGGGGUGGCGUUGCCCCACCGCACUGCUUGUGUGCGGUUCUCGCACGCUAGGGAUCCUAUCUUUGGUAAUCAGAUCAUUCCCGACACAGCAAUCCUGUCCGUGGUACCUUUUCAUCACGGUUUUGGCAUGUUCACGACUCUCGGCUAUUUGAUUUGCGGUUUCAGGGUCGUACUUAUGUAUCGGUUCGAGGAAGAACUGUUUUUGAGAUCCUUGCAAGAUUACAAGAUCCAGUCGGCCCUCCUUGUGCCAACGCUUUUCUCAUUCUUUGCGAAAUCGACACUUAUUGAUAAGUAUGACCUUUCCAAUCUGCAUGAGAUUGCCUCAGGGGGAGCGCCGCUUAGCAAGGAAGUCGGGGAGGCAGUGGCCAAGCGCUUCCACCUUCCCGGAAUUCGGCAGGGAUACGGGCUCACGGAGACAACAUCCGCGAUCCUUAUCACGCCCGAGGGUGACGAUAAGCCGGGAGCCGUCGGAAAAGUGGUCCCCUUCUUUGAAGCCAAGGUCGUAGACCUCGACACGGGAAAAACCCUCGGAGUGAACCAGAGGGGCGAGCUCUGCGUGAGAGGGCCGAUGAUCAUGUCAGGUUACGUGAAUAACCCUGAAGCGACGAAUGCGCUGAUCGACAAGGAUGGGUGGUUGCAUUCGGGAGACAUUGCCUAUUGGGAUGAGGAUGAGCACUUCUUUAUCGUAGAUCGACUUAAGAGCUUGAUCAAAUACAAAGGCUAUCAGGUAGCGCCUGCCGAGCUCGAGUCAAUCCUGCUCCAGCACCCCAACAUUUUCGACGCCGGAGUGGCCGGGUUGCCCGAUGACGACGCGGGUGAGCUGCCAGCGGCCGUGGUAGUCCUCGAACAUGGGAAAACAAUGACCGAAAAGGAGAUCGUGGACUACGUAGCAUCACAAGUGACGACUGCGAAGAAACUGAGGGGAGGGGUAGUCUUUGUGGACGAGGUCCCGAAAGGCUUGACUGGGAAGCUUGACGCUCGCAAAAUCCGGGAAAUCCUGAUUAAGGCAAAGAAAGGCGGGAAAAUCGCUGUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

In some embodiments, the polynucleotide of the disclosure comprises atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99% or about 100% identical to thepolynucleotide sequence set forth as SEQ ID NO: 65 in Table 5A, whereinthe protein encoded by the polynucleotide is capable of binding to thewild-type OX40 receptor.

In certain embodiments, the polynucleotide comprising an mRNA encodingan OX40L polypeptide of the present disclosure comprises (i) 5′ capprovided above, (ii) 5′ UTR, such as the sequences provided in Table 3above, (iii) an open reading frame encoding an OX40L polypeptide, suchas the sequences provided in Table 1 above, (iv) a stop codon, (v) a 3′UTR, such as the sequences provided in Table 4A or 4B above, and (vi) apoly-A tail provided above.

IV. METHODS OF MAKING POLYNUCLEOTIDES

The present disclosure also provides methods for making a polynucleotidedisclosed herein or a complement thereof. In some aspects, apolynucleotide (e.g., an mRNA) disclosed herein, and encoding anOX40Lpolypeptide, can be constructed using in vitro transcription. Inother aspects, a polynucleotide (e.g., an mRNA) disclosed herein, andencoding an OX40L polypeptide, can be constructed by chemical synthesisusing an oligonucleotide synthesizer. In other aspects, a polynucleotide(e.g., an mRNA) disclosed herein, and encoding an OX40L polypeptide ismade by using a host cell. In certain aspects, a polynucleotide (e.g.,an mRNA) disclosed herein, and encoding an OX40Lpolypeptide is made byone or more combination of the IVT, chemical synthesis, host cellexpression, or any other methods known in the art.

Naturally occurring nucleosides, non-naturally occurring nucleosides, orcombinations thereof, can totally or partially naturally replaceoccurring nucleosides present in the candidate nucleotide sequence andcan be incorporated into a sequence-optimized nucleotide sequence (e.g.,an mRNA) encoding an OX40L polypeptide. The resultant mRNAs can then beexamined for their ability to produce protein and/or produce atherapeutic outcome.

In Vitro Transcription-Enzymatic Synthesis

A polynucleotide disclosed herein can be transcribed using an in vitrotranscription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs can be selected from, but are notlimited to, those described herein including natural and unnatural(modified) NTPs. The polymerase can be selected from, but is not limitedto, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as,but not limited to, polymerases able to incorporate modified nucleicacids. See U.S. Publ. No. US20130259923.

The IVT system typically comprises a transcription buffer, nucleotidetriphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs canbe selected from, but are not limited to, those described hereinincluding natural and unnatural (modified) NTPs. The polymerase can beselected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate polynucleotides disclosed herein.

Any number of RNA polymerases or variants can be used in the synthesisof the polynucleotides of the present disclosure.

RNA polymerases can be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase is modified to exhibit an increased ability to incorporate a2′-modified nucleotide triphosphate compared to an unmodified RNApolymerase (see International Publication WO2008078180 and U.S. Pat. No.8,101,385).

Variants can be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants are evolved using the continuous directed evolutionsystem set out by Esvelt et al. (Nature (2011) 472(7344):499-503) whereclones of T7 RNA polymerase can encode at least one mutation such as,but not limited to, lysine at position 93 substituted for threonine(K93T), 14M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M2671, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants can encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112. Variants of RNApolymerase can also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one aspect, the polynucleotide can be designed to be recognized bythe wild type or variant RNA polymerases. In doing so, thepolynucleotide can be modified to contain sites or regions of sequencechanges from the wild type or parent chimeric polynucleotide.

Polynucleotide or nucleic acid synthesis reactions can be carried out byenzymatic methods utilizing polymerases. Polymerases catalyze thecreation of phosphodiester bonds between nucleotides in a polynucleotideor nucleic acid chain. Currently known DNA polymerases can be dividedinto different families based on amino acid sequence comparison andcrystal structure analysis. DNA polymerase I (pol I) or A polymerasefamily, including the Klenow fragments of E. Coli, Bacillus DNApolymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNAand DNA polymerases, is among the best studied of these families.Another large family is DNA polymerase α (pol α) or B polymerase family,including all eukaryotic replicating DNA polymerases and polymerasesfrom phages T4 and RB69. Although they employ similar catalyticmechanism, these families of polymerases differ in substratespecificity, substrate analog-incorporating efficiency, degree and ratefor primer extension, mode of DNA synthesis, exonuclease activity, andsensitivity against inhibitors.

DNA polymerases are also selected based on the optimum reactionconditions they require, such as reaction temperature, pH, and templateand primer concentrations. Sometimes a combination of more than one DNApolymerases is employed to achieve the desired DNA fragment size andsynthesis efficiency. For example, Cheng et al. increase pH, addglycerol and dimethyl sulfoxide, decrease denaturation times, increaseextension times, and utilize a secondary thermostable DNA polymerasethat possesses a 3′ to 5′ exonuclease activity to effectively amplifylong targets from cloned inserts and human genomic DNA. (Cheng et al.,PNAS, Vol. 91, 5695-5699 (1994)). RNA polymerases from bacteriophage T3,T7, and SP6 have been widely used to prepare RNAs for biochemical andbiophysical studies. RNA polymerases, capping enzymes, and poly-Apolymerases are disclosed in the co-pending International PublicationNo. WO2014028429.

In one aspect, the RNA polymerase which can be used in the synthesis ofthe polynucleotides described herein is a Syn5 RNA polymerase. (see Zhuet al. Nucleic Acids Research 2013). The Syn5 RNA polymerase wasrecently characterized from marine cyanophage Syn5 by Zhu et al. wherethey also identified the promoter sequence (see Zhu et al. Nucleic AcidsResearch 2013). Zhu et al. found that Syn5 RNA polymerase catalyzed RNAsynthesis over a wider range of temperatures and salinity as compared toT7 RNA polymerase. Additionally, the requirement for the initiatingnucleotide at the promoter was found to be less stringent for Syn5 RNApolymerase as compared to the T7 RNA polymerase making Syn5 RNApolymerase promising for RNA synthesis.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis of thepolynucleotides described herein. As a non-limiting example, a Syn5 RNApolymerase can be used in the synthesis of the polynucleotide requiringa precise 3′-termini.

In one aspect, a Syn5 promoter can be used in the synthesis of thepolynucleotides. As a non-limiting example, the Syn5 promoter can be5′-ATTGGGCACCCGTAAGGG-3′ as described by Zhu et al. (Nucleic AcidsResearch 2013).

In one aspect, a Syn5 RNA polymerase can be used in the synthesis ofpolynucleotides comprising at least one chemical modification describedherein and/or known in the art. (see e.g., the incorporation ofpseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research2013).

In one aspect, the polynucleotides described herein can be synthesizedusing a Syn5 RNA polymerase which has been purified using modified andimproved purification procedure described by Zhu et al. (Nucleic AcidsResearch 2013).

Various tools in genetic engineering are based on the enzymaticamplification of a target gene which acts as a template. For the studyof sequences of individual genes or specific regions of interest andother research needs, it is necessary to generate multiple copies of atarget gene from a small sample of polynucleotides or nucleic acids.Such methods can be applied in the manufacture of the polynucleotides ofthe disclosure.

Polymerase chain reaction (PCR) has wide applications in rapidamplification of a target gene, as well as genome mapping andsequencing. The key components for synthesizing DNA comprise target DNAmolecules as a template, primers complementary to the ends of target DNAstrands, deoxynucleoside triphosphates (dNTPs) as building blocks, and aDNA polymerase. As PCR progresses through denaturation, annealing andextension steps, the newly produced DNA molecules can act as a templatefor the next circle of replication, achieving exponentiallyamplification of the target DNA. PCR requires a cycle of heating andcooling for denaturation and annealing. Variations of the basic PCRinclude asymmetric PCR [Innis et al., PNAS, vol. 85, 9436-9440 (1988)],inverse PCR [Ochman et al., Genetics, vol. 120(3), 621-623, (1988)],reverse transcription PCR (RT-PCR) (Freeman et al., BioTechniques, vol.26(1), 112-22, 124-5 (1999)). In RT-PCR, a single stranded RNA is thedesired target and is converted to a double stranded DNA first byreverse transcriptase.

A variety of isothermal in vitro nucleic acid amplification techniqueshave been developed as alternatives or complements of PCR. For example,strand displacement amplification (SDA) is based on the ability of arestriction enzyme to form a nick. (Walker et al., PNAS, vol. 89,392-396 (1992), the contents of which are incorporated herein byreference in their entirety)). A restriction enzyme recognition sequenceis inserted into an annealed primer sequence. Primers are extended by aDNA polymerase and dNTPs to form a duplex. Only one strand of the duplexis cleaved by the restriction enzyme. Each single strand chain is thenavailable as a template for subsequent synthesis. SDA does not requirethe complicated temperature control cycle of PCR.

Nucleic acid sequence-based amplification (NASBA), also calledtranscription mediated amplification (TMA), is also an isothermalamplification method that utilizes a combination of DNA polymerase,reverse transcriptase, RNAse H, and T7 RNA polymerase. [Compton, Nature,vol. 350, 91-92 (1991)]. A target RNA is used as a template and areverse transcriptase synthesizes its complementary DNA strand. RNAse Hhydrolyzes the RNA template, making space for a DNA polymerase tosynthesize a DNA strand complementary to the first DNA strand which iscomplementary to the RNA target, forming a DNA duplex. T7 RNA polymerasecontinuously generates complementary RNA strands of this DNA duplex.These RNA strands act as templates for new cycles of DNA synthesis,resulting in amplification of the target gene.

Rolling-circle amplification (RCA) amplifies a single stranded circularpolynucleotide and involves numerous rounds of isothermal enzymaticsynthesis where D29 DNA polymerase extends a primer by continuouslyprogressing around the polynucleotide circle to replicate its sequenceover and over again. Therefore, a linear copy of the circular templateis achieved. A primer can then be annealed to this linear copy and itscomplementary chain can be synthesized. [See Lizardi et al., NatureGenetics, vol. 19, 225-232 (1998)]. A single stranded circular DNA canalso serve as a template for RNA synthesis in the presence of an RNApolymerase. (Daubendiek et al., JACS, vol. 117, 7818-7819 (1995)). Aninverse rapid amplification of cDNA ends (RACE) RCA is described byPolidoros et al. A messenger RNA (mRNA) is reverse transcribed intocDNA, followed by RNAse H treatment to separate the cDNA. The cDNA isthen circularized by CircLigase into a circular DNA. The amplificationof the resulting circular DNA is achieved with RCA. (Polidoros et al.,BioTechniques, vol. 41, 35-42 (2006)).

Any of the foregoing methods can be utilized in the manufacture of oneor more regions of the polynucleotides of the present disclosure.

Assembling polynucleotides or nucleic acids by a ligase is also widelyused. DNA or RNA ligases promote intermolecular ligation of the 5′ and3′ ends of polynucleotide chains through the formation of aphosphodiester bond. Ligase chain reaction (LCR) is a promisingdiagnosing technique based on the principle that two adjacentpolynucleotide probes hybridize to one strand of a target gene andcouple to each other by a ligase. If a target gene is not present, or ifthere is a mismatch at the target gene, such as a single-nucleotidepolymorphism (SNP), the probes cannot ligase. (Wiedmann et al., PCRMethods and Application, vol. 3 (4), s51-s64 (1994)). LCR can becombined with various amplification techniques to increase sensitivityof detection or to increase the amount of products if it is used insynthesizing polynucleotides and nucleic acids.

Several library preparation kits for nucleic acids are now commerciallyavailable. They include enzymes and buffers to convert a small amount ofnucleic acid samples into an indexed library for downstreamapplications. For example, DNA fragments can be placed in a NEBNEXT®ULTRA™ DNA Library Prep Kit by NEWENGLAND BIOLABS® for end preparation,ligation, size selection, clean-up, PCR amplification and finalclean-up.

Continued development is going on to improvement the amplificationtechniques. For example, U.S. Pat. No. 8,367,328 to Asada et al.,teaches utilizing a reaction enhancer to increase the efficiency of DNAsynthesis reactions by DNA polymerases. The reaction enhancer comprisesan acidic substance or cationic complexes of an acidic substance. U.S.Pat. No. 7,384,739 to Kitabayashi et al., teaches a carboxylateion-supplying substance that promotes enzymatic DNA synthesis, whereinthe carboxylate ion-supplying substance is selected from oxalic acid,malonic acid, esters of oxalic acid, esters of malonic acid, salts ofmalonic acid, and esters of maleic acid. U.S. Pat. No. 7,378,262 toSobek et al., discloses an enzyme composition to increase fidelity ofDNA amplifications. The composition comprises one enzyme with 3′exonuclease activity but no polymerase activity and another enzyme thatis a polymerase. Both of the enzymes are thermostable and are reversiblymodified to be inactive at lower temperatures.

U.S. Pat. No. 7,550,264 to Getts et al. teaches multiple round ofsynthesis of sense RNA molecules are performed by attachingoligodeoxynucleotides tails onto the 3′ end of cDNA molecules andinitiating RNA transcription using RNA polymerase. US Pat. PublicationNo. 2013/0183718 to Rohayem teaches RNA synthesis by RNA-dependent RNApolymerases (RdRp) displaying an RNA polymerase activity onsingle-stranded DNA templates. Oligonucleotides with non-standardnucleotides can be synthesized with enzymatic polymerization bycontacting a template comprising non-standard nucleotides with a mixtureof nucleotides that are complementary to the nucleotides of the templateas disclosed in U.S. Pat. No. 6,617,106 to Benner.

Chemical Synthesis

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an OX40L polypeptide. For example, a single DNA or RNAoligomer containing a codon-optimized nucleotide sequence coding for theparticular isolated polypeptide can be synthesized. In other aspects,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. In some aspects, theindividual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

A polynucleotide disclosed herein (e.g., mRNA) can be chemicallysynthesized using chemical synthesis methods and potential nucleobasesubstitutions known in the art. See, for example, InternationalPublication Nos. WO2014093924, WO2013052523; WO2013039857, WO2012135805,WO2013151671; U.S. Publ. No. US20130115272; or U.S. Pat. No. 8,999,380,U.S. Pat. No. 8,710,200.

Purification

Purification of the polynucleotides (e.g., mRNA) encoding an OX40Lpolypeptide described herein can include, but is not limited to,polynucleotide clean-up, quality assurance and quality control. Clean-upcan be performed by methods known in the arts such as, but not limitedto, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-Tbeads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) orHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedpolynucleotide” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

In some embodiments, purification of a polynucleotide (e.g., mRNA)encoding an OX40L polypeptide of the disclosure removes impurities thatcan reduce or remove an unwanted immune response, e.g., reducingcytokine activity.

In some embodiments, the polynucleotide (e.g., mRNA) encoding an OX40Lpolypeptide of the disclosure is purified prior to administration usingcolumn chromatography (e.g., strong anion exchange HPLC, weak anionexchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interactionHPLC (HIC-HPLC), or (LCMS)). In some embodiments, a columnchromatography (e.g., strong anion exchange HPLC, weak anion exchangeHPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), or (LCMS)) purified polynucleotide, which encodes an OX40Lpolypeptide disclosed herein increases expression of OX40L compared topolynucleotides encoding the OX40L polypeptide purified by a differentpurification method. In some embodiments, a column chromatography (e.g.,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS))purified polynucleotide encodes a mammalian OX40L polypeptide. In someembodiments, the purified polynucleotide encodes a murine OX40Lpolypeptide. In some embodiments, the purified polynucleotide encodes ahuman OX40L polypeptide. In some embodiments, the purifiedpolynucleotide encodes a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 1. In some embodiments, the purifiedpolynucleotide encodes a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 2.

In some embodiments, the purified polynucleotide is at least about 80%pure, at least about 85% pure, at least about 90% pure, at least about95% pure, at least about 96% pure, at least about 97% pure, at leastabout 98% pure, at least about 99% pure, or about 100% pure.

A quality assurance and/or quality control check can be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the polynucleotides can be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

V. MODIFICATIONS

As used herein in a polynucleotide comprising an mRNA encoding an OX40Lpolypeptide, the terms “chemical modification” or, as appropriate,“chemically modified” refer to modification with respect to adenosine(A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- ordeoxyribnucleosides in one or more of their position, pattern, percentor population. Generally, herein, these terms are not intended to referto the ribonucleotide modifications in naturally occurring 5′-terminalmRNA cap moieties.

In a polypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids.

The modifications can be various distinct modifications. In someembodiments, the regions can contain one, two, or more (optionallydifferent) nucleoside or nucleotide (nucleobase) modifications. In someembodiments, a modified polynucleotide, introduced to a cell can exhibitreduced degradation in the cell, as compared to an unmodifiedpolynucleotide. In other embodiments, the modification is in thenucleobase and/or the sugar structure. In yet other embodiments, themodification is in the backbone structure.

Chemical Modifications:

Some embodiments of the present disclosure provide a polynucleotidecomprising an mRNA encoding an OX40L polypeptide, wherein the mRNAincludes at least one chemical modification. In some embodiments, thechemical modification is selected from pseudouridine,N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methyluridine), 5-methoxyuridine, and2′-O-methyl uridine.

A “nucleoside” as used herein refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). A“nucleotide” as used herein refers to a nucleoside, including aphosphate group. Modified nucleotides can be synthesized by any usefulmethod, such as, for example, chemically, enzymatically, orrecombinantly, to include one or more modified or non-naturalnucleosides. Polynucleotides can comprise a region or regions of linkednucleosides. Such regions can have variable backbone linkages. Thelinkages can be standard phosphdioester linkages, in which case thepolynucleotides would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker can be incorporated into polynucleotides of thepresent disclosure.

The skilled artisan will appreciate that, except where otherwise noted,polynucleotide sequences set forth in the instant application willrecite “T”s in a representative DNA sequence but where the sequencerepresents RNA, the “T”s would be substituted for “U”s.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the polynucleotides of thepresent disclosure include, but are not limited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyl adenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine;N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine;1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate);Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6, N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycyti dine TP;2′-O-Methyl-5-(1-propynyl)cytidine TP; 3′-Ethynylcytidine TP;4′-Azidocytidine TP; 4′-Carbocyclic cytidine TP; 4′-Ethynylcytidine TP;5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP;5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-CyanocytidineTP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5′-Homo-cytidine TP;5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidineTP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine;N2,2′-O-dimethylguanosine; N2-methylguanosine; Wyosine;1,2′-O-dimethylguanosine; 1-methylguanosine; 2′-O-methylguanosine;2′-O-ribosylguanosine (phosphate); 2′-O-methylguanosine;2′-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine;7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine;N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carb amoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methyluridine), 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP;5-Oxyacetic acid-methyl ester-Uridine TP; N1-methyl-pseudo-uridine;uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylaminocarbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2(thio)pseudouracil; 2′ deoxy uridine; 2′ fluorouridine; 2-(thio)uracil;2,4-(dithio)psuedouracil; 2′ methyl, 2′amino, 2′azido,2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP;2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxy uridine; 2′fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridineTP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridineTP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridineTP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy} ]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide includes a combination of at least two (e.g., 2, 3, 4or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in the polynucleotidecomprising an mRNA encoding an OX40L polypeptide are selected from thegroup consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ),2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methyluridine), 5-methoxyuridine and 2′-O-methyluridine. In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) includes a combination ofat least two (e.g., 2, 3, 4 or more) of the aforementioned modifiednucleobases.

In some embodiments, modified nucleobases in the polynucleotidecomprising an mRNA encoding an OX40L polypeptide are selected from thegroup consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine(mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine andα-thio-adenosine. In some embodiments, the polynucleotide includes acombination of at least two (e.g., 2, 3, 4 or more) of theaforementioned modified nucleobases.

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide comprises pseudouridine (ψ) and 5-methyl-cytidine(m5C). In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotidecomprising an mRNA encoding an OX40L polypeptide comprises1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide comprising an mRNA encoding an OX40Lpolypeptide comprises 2-thiouridine (s2U). In some embodiments, thepolynucleotide comprising an mRNA encoding an OX40L polypeptidecomprises 2-thiouridine and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide comprising an mRNA encoding an OX40Lpolypeptide comprises methoxy-uridine (mo5U). In some embodiments, thepolynucleotide comprising an mRNA encoding an OX40L polypeptidecomprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 2′-O-methyl uridine. In some embodiments, thepolynucleotide comprising an mRNA encoding an OX40L polypeptidecomprises 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide comprising an mRNA encoding an OX40Lpolypeptide comprises N6-methyl-adenosine (m6A). In some embodiments,the polynucleotide comprising an mRNA encoding an OX40L polypeptidecomprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide is uniformly modified (e.g., fully modified, modifiedthroughout the entire sequence) for a particular modification. Forexample, a polynucleotide can be uniformly modified with5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m5C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the modified nucleobase is a modified cytosine.Examples of nucleobases and nucleosides having a modified cytosineinclude N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Example nucleobases and nucleosides having a modified uridine include5-cyano uridine or 4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Example nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A),N6-methyl-adenosine (m6A), and 2,6-Diaminopurine.

In some embodiments, a modified nucleobase is a modified guanine.Example nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methylinosine (m1I), wyosine (imG), methylwyosine (mimG),7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, or 7-methyl-8-oxo-guanosine.

Other modifications which can be useful in the polynucleotidescomprising an mRNA encoding an OX40L polypeptide of the presentdisclosure are listed in Table 6.

TABLE 6 Additional Modification types Name Type 2,6-(diamino)purineOther 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other1,3-(diaza)-2-(oxo)-phenthiazin-1-yl Other1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other1,3,5-(triaza)-2,6-(dioxa)-naphthalene Other 2(amino)purine Other2,4,5-(trimethyl)phenyl Other 2′methyl, 2′amino, 2′azido,2′fluro-cytidine Other 2′methyl, 2′amino, 2′azido, 2′fluro-adenine Other2′methyl, 2′amino, 2′azido, 2′fluro-uridine Other2′-amino-2′-deoxyribose Other 2-amino-6-Chloro-purine Other2-aza-inosinyl Other 2′-azido-2′-deoxyribose Other2′fluoro-2′-deoxyribose Other 2′-fluoro-modified bases Other2′-O-methyl-ribose Other 2-oxo-7-aminopyridopyrimidin-3-yl Other2-oxo-pyridopyrimidine-3-yl Other 2-pyridinone Other 3 nitropyrroleOther 3-(methyl)-7-(propynyl)isocarbostyrilyl Other3-(methyl)isocarbostyrilyl Other 4-(fluoro)-6-(methyl)benzimidazoleOther 4-(methyl)benzimidazole Other 4-(methyl)indolyl Other4,6-(dimethyl)indolyl Other 5 nitroindole Other 5 substitutedpyrimidines Other 5-(methyl)isocarbostyrilyl Other 5-nitroindole Other6-(aza)pyrimidine Other 6-(azo)thymine Other 6-(methyl)-7-(aza)indolylOther 6-chloro-purine Other 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Other7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl Other7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl Other7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(aza)indolyl Other7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl Other-yl 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-Other 1-yl7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin- Other1-yl 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-ylOther 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-ylOther 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-ylOther 7-(propynyl)isocarbostyrilyl Other 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl Other 7-deaza-inosinyl Other 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other 9-(methyl)-imidizopyridinylOther aminoindolyl Other anthracenyl Otherbis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on- Other3-yl bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Otherdifluorotolyl Other hypoxanthine Other imidizopyridinyl Other inosinylOther isocarbostyrilyl Other isoguanisine Other N2-substituted purinesOther N6-methyl-2-amino-purine Other N6-substituted purines OtherN-alkylated derivative Other napthalenyl Other nitrobenzimidazolyl Othernitroimidazolyl Other nitroindazolyl Other nitropyrazolyl OtherNubularine Other O6-substituted purines Other O-alkylated derivativeOther ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on- Other3-yl ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl OtherOxoformycin TP Otherpara-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on- Other 3-ylpara-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Other pentacenylOther phenanthracenyl Other phenyl Other propynyl-7-(aza)indolyl Otherpyrenyl Other pyridopyrimidin-3-yl Other pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl Other pyrrolo-pyrimidin-2-on-3-ylOther pyrrolopyrimidinyl Other pyrrolopyrizinyl Other stilbenzyl Othersubstituted 1,2,4-triazoles Other tetracenyl Other tubercidine Otherxanthine Other Xanthosine-5′-TP Other 2-thio-zebularine Other5-aza-2-thio-zebularine Other 7-deaza-2-amino-purine Other pyridin-4-oneribonucleoside Other 2-Amino-riboside-TP Other Formycin A TP OtherFormycin B TP Other Pyrrolosine TP Other 2′-OH-ara-adenosine TP Other2′-OH-ara-cytidine TP Other 2′-OH-ara-uridine TP Other2′-OH-ara-guanosine TP Other 5-(2-carbomethoxyvinyl)uridine TP OtherN6-(19-Amino-pentaoxanonadecyl)adenosine TP Other

The polynucleotides comprising an mRNA encoding an OX40L polypeptide ofthe present disclosure can include any useful linker between thenucleosides. Such linkers, including backbone modifications are given inTable 7.

TABLE 7 Linker modifications Name TYPE 3′-alkylene phosphonates Linker3′-amino phosphoramidate Linker alkene containing backbones Linkeraminoalkylphosphoramidates Linker aminoalkylphosphotriesters Linkerboranophosphates Linker —CH2-0-N(CH3)—CH2— Linker—CH2—N(CH3)—N(CH3)—CH2— Linker —CH2—NH—CH2— Linker chiral phosphonatesLinker chiral phosphorothioates Linker formacetyl and thioformacetylbackbones Linker methylene (methylimino) Linker methylene formacetyl andthioformacetyl backbones Linker methyleneimino and methylenehydrazinobackbones Linker morpholino linkages Linker —N(CH3)—CH2—CH2— Linkeroligonucleosides with heteroatom internucleoside linkage Linkerphosphinates Linker phosphoramidates Linker phosphorodithioates Linkerphosphorothioate internucleoside linkages Linker phosphorothioatesLinker phosphotriesters Linker PNA Linker siloxane backbones Linkersulfamate backbones Linker sulfide sulfoxide and sulfone backbonesLinker sulfonate and sulfonamide backbones Linkerthionoalkylphosphonates Linker thionoalkylphosphotriesters Linkerthionophosphoramidates Linker

The polynucleotide comprising an mRNA encoding an OX40L polypeptide ofthe present disclosure can include any useful modification, such as tothe sugar, the nucleobase, or the internucleoside linkage (e.g. to alinking phosphate/to a phosphodiester linkage/to the phosphodiesterbackbone). One or more atoms of a pyrimidine nucleobase can be replacedor substituted with optionally substituted amino, optionally substitutedthiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo(e.g., chloro or fluoro). In certain embodiments, modifications (e.g.,one or more modifications) are present in each of the sugar and theinternucleoside linkage. Modifications according to the presentdisclosure can be modifications of ribonucleic acids (RNAs) todeoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs), hexitol nucleic acids (HNAs), or hybrids thereof. Additionalmodifications are described herein. Modified nucleic acids and theirsynthesis are disclosed in co-pending International Patent PublicationNo. WO2013052523.

In some embodiments, the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure does not substantiallyinduce an innate immune response of a cell into which the mRNA isintroduced. Features of an induced innate immune response include 1)increased expression of pro-inflammatory cytokines, 2) activation ofintracellular PRRs (RIG-I, MDAS, etc, and/or 3) termination or reductionin protein translation.

Any of the regions of the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure can be chemically modifiedas taught herein or as taught in International Application PublicationNumber WO2013/052523 A1.

In some embodiments, a modified polynucleotide, e.g., mRNA comprising atleast one modification described herein, of the disclosure encodes anOX40L polypeptide. In some embodiments, the modified polynucleotide,e.g., mRNA comprising at least one modification described herein, of thedisclosure encodes a human OX40L polypeptide. In some embodiments, themodified polynucleotide, e.g., mRNA comprising at least one modificationdescribed herein, of the disclosure encodes a polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 1. In some embodiments, themodified polynucleotide, e.g., mRNA comprising at least one modificationdescribed herein, of the disclosure encodes a polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 2. In some embodiments, themodified polynucleotide, e.g., mRNA comprising at least one modificationdescribed herein, of the disclosure comprises the sequence set forth inSEQ ID NO: 65.

In some embodiments, the modified polynucleotide, e.g., mRNA comprisingat least one modification described herein, of the disclosure encodes atleast one OX40L mutant, a fragment, or variant thereof, e.g., an OX40Lfunctional fragment comprising the extracellular domain of OX40L or afragment of the extracellular domain of OX40L, the cytoplasmic domain ofOX40L or a fragment of the cytoplasmic domain of OX40L, and/or thetransmembrane domain of OX40L or a fragment of the transmembrane domainof OX40L.

In some embodiments, the modified polynucleotide, e.g., mRNA comprisingat least one modification described herein, of the disclosure isselected from the OX40L nucleic acid sequences listed in Table 1 (e.g.,selected from SEQ ID NOs: 4-21).

The polynucleotide comprising an mRNA encoding an OX40L polypeptide ofthe present disclosure can also include building blocks, e.g., modifiedribonucleosides, and modified ribonucleotides, of polynucleotidemolecules. For example, these building blocks can be useful forpreparing the polynucleotides of the disclosure. Such building blocksare taught in International Patent Publication No. WO2013052523 andInternational Application Publication No. WO 2014/093924 A1.

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which can be incorporated into a polynucleotide (e.g., RNAor mRNA, as described herein) comprising an mRNA encoding an OX40Lpolypeptide of the present disclosure, can be modified on the sugar ofthe ribonucleic acid. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different substituents. Exemplarysubstitutions at the 2′-position include, but are not limited to, H,halo, optionally substituted C₁₋₆ alkyl; optionally substituted C₁₋₆alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionally substitutedC₃₋₈ cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy; optionallysubstituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀ aryl-C₁₋₆alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; a sugar (e.g.,ribose, pentose, or any described herein); a polyethyleneglycol (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H or optionally substituted alkyl,and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16,from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connectedby a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of thesame ribose sugar, where exemplary bridges included methylene,propylene, ether, or amino bridges; aminoalkyl, as defined herein;aminoalkoxy, as defined herein; amino as defined herein; and amino acid,as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar. Suchsugar modifications are taught International Patent Publication No.WO2013052523 and International Patent Application No. PCT/US2013/75177.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides comprising an mRNA encoding an OX40L polypeptide ofthe disclosure can include a combination of modifications to the sugar,the nucleobase, and/or the internucleoside linkage. These combinationscan include any one or more modifications described herein.

Examples of modified nucleotides and modified nucleotide combinationsare provided below in Table 8. These combinations of modifiednucleotides can be used to form the polynucleotides of the disclosure.Unless otherwise noted, the modified nucleotides can be completelysubstituted for the natural nucleotides of the polynucleotides of thedisclosure. As a non-limiting example, the natural nucleotide uridinecan be substituted with a modified nucleoside described herein. Inanother non-limiting example, the natural nucleotide uridine can bepartially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99.9%) with at least one of the modified nucleoside disclosed herein.Any combination of base/sugar or linker can be incorporated into thepolynucleotides of the disclosure and such modifications are taught inInternational Patent Publication Nos. WO 2013/052523 and WO 2014/093924A1.

TABLE 8 Combinations Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudouridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1- methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/ about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

Additional examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 9.

TABLE 9 Additional combinations Uracil Cytosine Adenine Guanine5-methoxy-UTP CTP ATP GTP 5-Methoxy-UTP N4Ac-CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP 5-Trifluoromethyl-CTP ATP GTP5-Methoxy-UTP 5-Hydroxymethyl-CTP ATP GTP 5-Methoxy-UTP 5-Bromo-CTP ATPGTP 5-Methoxy-UTP N4Ac-CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 5-Trifluoromethyl-CTPATP GTP 5-Methoxy-UTP 5-Hydroxymethyl-CTP ATP GTP 5-Methoxy-UTP5-Bromo-CTP ATP GTP 5-Methoxy-UTP N4—Ac-CTP ATP GTP 5-Methoxy-UTP5-Iodo-CTP ATP GTP 5-Methoxy-UTP 5-Bromo-CTP ATP GTP 5-Methoxy-UTP CTPATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP5-Methyl-CTP ATP GTP 5-Methoxy-UTP CTP Alpha-thio-ATP GTP 5-Methoxy-UTP5-Methyl-CTP Alpha-thio-ATP GTP 5-Methoxy-UTP CTP ATP Alpha-thio-GTP5-Methoxy-UTP 5-Methyl-CTP ATP Alpha-thio-GTP 5-Methoxy-UTP CTPN6—Me-ATP GTP 5-Methoxy-UTP 5-Methyl-CTP N6—Me-ATP GTP 5-Methoxy-UTP CTPATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP 5-Ethyl-CTPATP GTP 5-Methoxy-UTP 5-Methoxy-CTP ATP GTP 5-Methoxy-UTP 5-Ethynyl-CTPATP GTP 5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75%5-Methoxy-UTP + 25% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP 50%5-Methoxy-UTP + 50% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP 25%5-Methoxy-UTP + 75% 1- 5-Methyl-CTP ATP GTP Methyl-pseudo-UTP5-Methoxy-UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50%5-Methyl-CTP + 50% CTP ATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTPATP GTP 75% 5-Methoxy-UTP + 25% 1- 75% 5-Methyl-CTP + 25% CTP ATP GTPMethyl-pseudo-UTP 75% 5-Methoxy-UTP + 25% 1- 50% 5-Methyl-CTP + 50% CTPATP GTP Methyl-pseudo-UTP 75% 5-Methoxy-UTP + 25% 1- 25% 5-Methyl-CTP +75% CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50% 1- 75%5-Methyl-CTP + 25% CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50%1- 50% 5-Methyl-CTP + 50% CTP ATP GTP Methyl-pseudo-UTP 50%5-Methoxy-UTP + 50% 1- 25% 5-Methyl-CTP + 75% CTP ATP GTPMethyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 75% 5-Methyl-CTP + 25% CTPATP GTP Methyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 50% 5-Methyl-CTP +50% CTP ATP GTP Methyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- 25%5-Methyl-CTP + 75% CTP ATP GTP Methyl-pseudo-UTP 75% 5-Methoxy-UTP + 25%1- CTP ATP GTP Methyl-pseudo-UTP 50% 5-Methoxy-UTP + 50% 1- CTP ATP GTPMethyl-pseudo-UTP 25% 5-Methoxy-UTP + 75% 1- CTP ATP GTPMethyl-pseudo-UTP 5-methoxy-UTP (In House) CTP ATP GTP 5-methoxy-UTP(Hongene) CTP ATP GTP 5-methoxy-UTP (Hongene) 5-Methyl-CTP ATP GTP5-Methoxy-UTP CTP ATP GTP 5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP5-Methyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP5-Methoxy-UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50%5-Methyl-CTP + 50% CTP ATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTPATP GTP 75% 5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP75% 5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP5-Methoxy-UTP 5-Methyl-CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP5-Methyl-CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP ATP GTP 5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTPATP GTP 5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 50%5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP CTP ATP GTP 50% 5-Methoxy-UTP + 50% UTP CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP CTP ATP GTP 5-Methoxy-UTP CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTPCTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP ATPGTP 25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Methyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Fluoro-CTP ATP GTP 5-Methoxy-UTP 5-Phenyl-CTP ATP GTP 5-Methoxy-UTPN4-Bz-CTP ATP GTP 5-Methoxy-UTP CTP N6-Isopentenyl- GTP ATP5-Methoxy-UTP N4—Ac-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%N4—Ac-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% N4—Ac-CTP +25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25% N4—Ac-CTP + 75% CTP ATPGTP 75% 5-Methoxy-UTP + 25% UTP 75% N4—Ac-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Hydroxymethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP25% 5-Hydroxymethyl-CTP + 75% ATP GTP CTP 25% 5-Methoxy-UTP + 75% UTP75% 5-Hydroxymethyl-CTP + 25% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP25% 5-Hydroxymethyl-CTP + 75% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP75% 5-Hydroxymethyl-CTP + 25% ATP GTP CTP 5-Methoxy-UTP N4-Methyl CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% N4-Methyl CTP + 75% CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% N4-Methyl CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% N4-Methyl CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% N4-Methyl CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Trifluoromethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP25% 5-Trifluoromethyl-CTP + 75% ATP GTP CTP 25% 5-Methoxy-UTP + 75% UTP75% 5-Trifluoromethyl-CTP + 25% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP25% 5-Trifluoromethyl-CTP + 75% ATP GTP CTP 75% 5-Methoxy-UTP + 25% UTP75% 5-Trifluoromethyl-CTP + 25% ATP GTP CTP 5-Methoxy-UTP 5-Bromo-CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP + 75% CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 75% 5-Bromo-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Iodo-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Iodo-CTP + 75% CTPATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Iodo-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Iodo-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Iodo-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Ethyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethyl-CTP + 75%CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP + 25% CTP ATPGTP 75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Ethyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP5-Methoxy-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25% 5-Methoxy-CTP +75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75% 5-Methoxy-CTP + 25% CTPATP GTP 75% 5-Methoxy-UTP + 25% UTP 25% 5-Methoxy-CTP + 75% CTP ATP GTP75% 5-Methoxy-UTP + 25% UTP 75% 5-Methoxy-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Ethynyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%5-Ethynyl-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75%5-Ethynyl-CTP + 25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25%5-Ethynyl-CTP + 75% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 75%5-Ethynyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 5-Pseudo-iso-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 25% 5-Pseudo-iso-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Pseudo-iso-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Pseudo-iso-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Pseudo-iso-CTP + 25% CTP ATP GTP5-Methoxy-UTP 5-Formyl-CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 25%5-Formyl-CTP + 75% CTP ATP GTP 25% 5-Methoxy-UTP + 75% UTP 75%5-Formyl-CTP + 25% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 25%5-Formyl-CTP + 75% CTP ATP GTP 75% 5-Methoxy-UTP + 25% UTP 75%5-Formyl-CTP + 25% CTP ATP GTP 5-Methoxy-UTP 5-Aminoallyl-CTP ATP GTP25% 5-Methoxy-UTP + 75% UTP 25% 5-Aminoallyl-CTP + 75% CTP ATP GTP 25%5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 25% 5-Aminoallyl-CTP + 75% CTP ATP GTP 75%5-Methoxy-UTP + 25% UTP 75% 5-Aminoallyl-CTP + 25% CTP ATP GTP

VI. PHARMACEUTICAL COMPOSITIONS Formulation, Administration, Deliveryand Dosing

The present disclosure provides use of a pharmaceutical compositioncomprising a polynucleotide which comprises an mRNA encoding an OX40Lpolypeptide to activate T cells in a subject in need thereof. Thepresent disclosure further provides use of a pharmaceutical compositioncomprising a polynucleotide which comprises an mRNA encoding an OX40Lpolypeptide to increase the number of Natural Killer (NK) cells in asubject in need thereof. In some embodiments of the disclosure, thepolynucleotide is formulated in compositions and complexes incombination with one or more pharmaceutically acceptable excipients.Pharmaceutical compositions can optionally comprise one or moreadditional active substances, e.g. therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent disclosure can be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents can be found, for example, in Remington: The Science and Practiceof Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides to bedelivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals.

In some embodiments, the polynucleotide of the present disclosure isformulated for subcutaneous, intravenous, intraperitoneal, intratumoral,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial,intraventricular, oral, inhalation spray, topical, rectal, nasal,buccal, vaginal, intratumoral, or implanted reservoir intramuscular,subcutaneous, intratumoral, or intradermal delivery. In otherembodiments, the polynucleotide is formulated for intratumoral,intraperitoneal, or intravenous delivery. In a particular embodiment,the polynucleotide of the present disclosure is formulated forintratumoral delivery.

Formulations of the pharmaceutical compositions described herein can beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition can comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The polynucleotides comprising an mRNA encoding an OX40L polypeptide ofthe disclosure can be formulated using one or more excipients to: (1)increase stability; (2) increase cell transfection; (3) permit thesustained or delayed release (e.g., from a depot formulation of thepolynucleotide); (4) alter the biodistribution (e.g., target thepolynucleotide to specific tissues or cell types); (5) increase thetranslation of encoded protein in vivo; and/or (6) alter the releaseprofile of encoded protein in vivo. In addition to traditionalexcipients such as any and all solvents, dispersion media, diluents, orother liquid vehicles, dispersion or suspension aids, surface activeagents, isotonic agents, thickening or emulsifying agents,preservatives, excipients of the present disclosure can include, withoutlimitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotides (e.g., for transplantation into asubject), hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the disclosure can include one or moreexcipients, each in an amount that together increases the stability ofthe polynucleotide, increases cell transfection by the polynucleotide,increases the expression of polynucleotides encoded protein, and/oralters the release profile of polynucleotide encoded proteins. Further,the polynucleotides of the present disclosure can be formulated usingself-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein can beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosurecan be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure canvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition cancomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition can comprise between 0.1% and 100%, e.g.,between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In some embodiments, the formulations described herein contain at leastone polynucleotide. As a non-limiting example, the formulations contain1, 2, 3, 4 or 5 polynucleotides. In other embodiments, thepolynucleotide of the disclosure is formulated for intratumoral deliveryin a tumor of a patient in need thereof.

Pharmaceutical formulations can additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006). The use of aconventional excipient medium can be contemplated within the scope ofthe present disclosure, except insofar as any conventional excipientmedium can be incompatible with a substance or its derivatives, such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition.

In some embodiments, the particle size of the lipid nanoparticle isincreased and/or decreased. The change in particle size can be able tohelp counter biological reaction such as, but not limited to,inflammation or can increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients canoptionally be included in the pharmaceutical formulations of thedisclosure.

In some embodiments, the polynucleotides is administered in or with,formulated in or delivered with nanostructures that can sequestermolecules such as cholesterol. Non-limiting examples of thesenanostructures and methods of making these nanostructures are describedin US Patent Publication No. US20130195759. Exemplary structures ofthese nanostructures are shown in US Patent Publication No.US20130195759, and can include a core and a shell surrounding the core.

Lipidoids

The polynucleotide comprising an mRNA encoding an OX40L polypeptide canbe formulated with lipidoids. The synthesis of lipidoids has beenextensively described and formulations containing these compounds areparticularly suited for delivery of polynucleotides (see Mahon et al.,Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al.,Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc NatlAcad Sci US A. 2011 108:12996-3001).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010), the present disclosure describes their formulation anduse in delivering polynucleotides.

Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, as judged by the production of an encoded protein,following the injection of a lipidoid formulation via localized and/orsystemic routes of administration. Lipidoid complexes of polynucleotidescan be administered by various means including, but not limited to,intravenous, intraperitoneal, intratumoral, intramuscular, orsubcutaneous routes.

In vivo delivery of nucleic acids can be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, polynucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol Ther. 2009 17:872-879). As an example, small changesin the anchor chain length of poly(ethylene glycol) (PEG) lipids canresult in significant effects on in vivo efficacy. Formulations with thedifferent lipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010)), C12-200 (including derivatives and variants), and MD 1,can be tested for in vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotides.

Lipidoids and polynucleotide formulations comprising lipidoids aredescribed in International Patent Application No. PCT/US2014/097077.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotides of the disclosure can be formulated using one ormore liposomes, lipoplexes, or lipid nanoparticles. In one embodiment,pharmaceutical compositions of the polynucleotides comprising an mRNAencoding an OX40L polypeptide include liposomes. Liposomes areartificially-prepared vesicles which can primarily be composed of alipid bilayer and can be used as a delivery vehicle for theadministration of pharmaceutical formulations. Liposomes can be ofdifferent sizes such as, but not limited to, a multilamellar vesicle(MLV) which can be hundreds of nanometers in diameter and can contain aseries of concentric bilayers separated by narrow aqueous compartments,a small unicellular vesicle (SUV) which can be smaller than 50 nm indiameter, and a large unilamellar vesicle (LUV) which can be between 50and 500 nm in diameter. Liposome design can include, but is not limitedto, opsonins or ligands in order to improve the attachment of liposomesto unhealthy tissue or to activate events such as, but not limited to,endocytosis. Liposomes can contain a low or a high pH in order toimprove the delivery of the pharmaceutical formulations.

The formation of liposomes can depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

As a non-limiting example, liposomes such as synthetic membrane vesiclesare prepared by the methods, apparatus and devices described in USPatent Publication No. US20130177638, US20130177637, US20130177636,US20130177635, US20130177634, US20130177633, US20130183375,US20130183373 and US20130183372.

In one embodiment, pharmaceutical compositions described herein include,without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (as described in US20100324120) and liposomes which can deliversmall molecule drugs such as, but not limited to, DOXIL® from JanssenBiotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein caninclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104). The original manufacturemethod by Wheeler et al. was a detergent dialysis method, which waslater improved by Jeffs et al. and is referred to as the spontaneousvesicle formation method. The liposome formulations are composed of 3 to4 lipid components in addition to the polynucleotide. As an example aliposome can contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations contain, butare not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations comprise from about 25.0%cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol toabout 45.0% cholesterol, from about 35.0% cholesterol to about 50.0%cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. In other embodiments, formulations comprise a percentage ofcholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%,36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulationscomprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% toabout 15.0% DSPC.

In one embodiment, pharmaceutical compositions include liposomes whichare formed to deliver polynucleotides comprising an mRNA encoding anOX40L polypeptide. The polynucleotides can be encapsulated by theliposome and/or it can be contained in an aqueous core which can then beencapsulated by the liposome (see International Pub. Nos. WO2012031046,WO2012031043, WO2012030901 and WO2012006378 and US Patent PublicationNo. US20130189351, US20130195969 and US20130202684).

In another embodiment, liposomes are formulated for targeted delivery.As a non-limiting example, the liposome is formulated for targeteddelivery to the liver. The liposome used for targeted delivery caninclude, but is not limited to, the liposomes described in and methodsof making liposomes described in US Patent Publication No.US20130195967.

In another embodiment, the polynucleotides comprising an mRNA encodingan OX40L polypeptide is formulated in a cationic oil-in-water emulsionwhere the emulsion particle comprises an oil core and a cationic lipidwhich can interact with the polynucleotide anchoring the molecule to theemulsion particle (see International Pub. No. WO2012006380).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide is formulated in a water-in-oil emulsion comprising acontinuous hydrophobic phase in which the hydrophilic phase isdispersed. As a non-limiting example, the emulsion can be made by themethods described in International Publication No. WO201087791.

In another embodiment, the lipid formulation includes at least cationiclipid, a lipid which can enhance transfection and a least one lipidwhich contains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582). Inanother embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide is formulated in a lipid vesicle which can havecrosslinks between functionalized lipid bilayers (see U.S. Pub. No.20120177724).

In one embodiment, the polynucleotides are formulated in a liposome asdescribed in International Patent Publication No. WO2013086526. Thepolynucleotides can be encapsulated in a liposome using reverse pHgradients and/or optimized internal buffer compositions as described inInternational Patent Publication No. WO2013086526.

In one embodiment, the polynucleotide pharmaceutical compositions areformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes(Quiet Therapeutics, Israel).

In one embodiment, the cationic lipid is a low molecular weight cationiclipid such as those described in US Patent Application No. 20130090372.In another embodiment, the polynucleotides comprising an mRNA encodingan OX40L polypeptide are formulated in a lipid vesicle which can havecrosslinks between functionalized lipid bilayers.

In other embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a liposome comprising a cationiclipid. The liposome can have a molar ratio of nitrogen atoms in thecationic lipid to the phosphates in the polynucleotide (N:P ratio) ofbetween 1:1 and 20:1 as described in International Publication No.WO2013006825. In another embodiment, the liposome can have a N:P ratioof greater than 20:1 or less than 1:1.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a lipid-polycation complex. Theformation of the lipid-polycation complex can be accomplished by methodsknown in the art and/or as described in U.S. Pub. No. 20120178702. As anon-limiting example, the polycation includes a cationic peptide or apolypeptide such as, but not limited to, polylysine, polyornithineand/or polyarginine and the cationic peptides described in InternationalPub. No. WO2012013326 or US Patent Pub. No. US20130142818. In anotherembodiment, the polynucleotides are formulated in a lipid-polycationcomplex which can further include a non-cationic lipid such as, but notlimited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in an aminoalcohol lipidoid.Aminoalcohol lipidoids which can be used in the present disclosure canbe prepared by the methods described in U.S. Pat. No. 8,450,298.

The liposome formulation can be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176), the liposomeformulation was composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid couldmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200). In some embodiments,liposome formulations comprise from about 35 to about 45% cationiclipid, from about 40% to about 50% cationic lipid, from about 50% toabout 60% cationic lipid and/or from about 55% to about 65% cationiclipid. In some embodiments, the ratio of lipid to mRNA in liposomes isfrom about 5:1 to about 20:1, from about 10:1 to about 25:1, from about15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations is increased or decreased and/or the carbon chain length ofthe PEG lipid is modified from C14 to C18 to alter the pharmacokineticsand/or biodistribution of the LNP formulations. As a non-limitingexample, LNP formulations contain from about 0.5% to about 3.0%, fromabout 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0%to about 4.5%, from about 2.5% to about 5.0% and/or from about 3.0% toabout 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In another embodiment the PEG-c-DOMG can bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid can be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a lipid nanoparticle such as thosedescribed in International Publication No. WO2012170930.

In another embodiment, the formulation comprising the polynucleotide isa nanoparticle which can comprise at least one lipid. The lipid can beselected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids and amino alcohol lipids. In another aspect, the lipidis a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA,DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The aminoalcohol cationic lipid can be the lipids described in and/or made by themethods described in US Patent Publication No. US20130150625. As anon-limiting example, the cationic lipid can be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

In some embodiments, the polynucleotide of the disclosure is formulatedin a lipid nanoparticle, wherein the polynucleotide comprises an mRNAencoding a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotide ofthe disclosure is formulated in a lipid nanoparticle, wherein thepolynucleotide comprises the sequence set forth in SEQ ID NO: 65.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In one embodiment, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the formulation includes from about 25% to about 75%on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In one embodiment, the formulation includes from about 0.5% to about 15%on a molar basis of the neutral lipid e.g., from about 3 to about 12%,from about 5 to about 10% or about 15%, about 10%, or about 7.5% on amolar basis. Exemplary neutral lipids include, but are not limited to,DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In one embodiment, the formulation includes from about 0.5%to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g.,about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%,about 1.5%, about 3.5%, or about 5% on a molar basis. In one embodiment,the PEG or PEG modified lipid comprises a PEG molecule of an averagemolecular weight of 2,000 Da. In other embodiments, the PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof less than 2,000, for example around 1,500 Da, around 1,000 Da, oraround 500 Da. Exemplary PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J.Controlled Release, 107, 276-287 (2005).

In one embodiment, the formulations of the disclosures include 25-75% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include 35-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include 45-65% ofa cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include about 60%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include about 50%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In one embodiment, the formulations of the disclosures include about 40%of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include about57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In one embodiment, the formulations of the disclosures include about57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005)), about 7.5% of the neutral lipid, about 31.5% ofthe sterol, and about 3.5% of the PEG or PEG-modified lipid on a molarbasis.

In some embodiments, lipid nanoparticle formulation consists essentiallyof a lipid mixture in molar ratios of about 20-70% cationic lipid: 5-45%neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid; e.g., ina molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid:25-55% cholesterol: 0.5-15% PEG-modified lipid.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary lipid nanoparticle compositions and methods of making same aredescribed, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578.

In one embodiment, the lipid nanoparticle formulations described hereincomprise a cationic lipid, a PEG lipid and a structural lipid andoptionally comprise a non-cationic lipid. As a non-limiting example, thelipid nanoparticle comprises about 40-60% of cationic lipid, about 5-15%of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of astructural lipid. As another non-limiting example, the lipidnanoparticle comprises about 50% cationic lipid, about 10% non-cationiclipid, about 1.5% PEG lipid and about 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle comprises about 55%cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid andabout 32.5% structural lipid. In one embodiment, the cationic lipid isany cationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In one embodiment, the lipid nanoparticle formulations described hereinis 4 component lipid nanoparticles. The lipid nanoparticle can comprisea cationic lipid, a non-cationic lipid, a PEG lipid and a structurallipid. As a non-limiting example, the lipid nanoparticle can compriseabout 40-60% of cationic lipid, about 5-15% of a non-cationic lipid,about 1-2% of a PEG lipid and about 30-50% of a structural lipid. Asanother non-limiting example, the lipid nanoparticle can comprise about50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipidand about 38.5% structural lipid. As yet another non-limiting example,the lipid nanoparticle can comprise about 55% cationic lipid, about 10%non-cationic lipid, about 2.5% PEG lipid and about 32.5% structurallipid. In one embodiment, the cationic lipid can be any cationic lipiddescribed herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMAand L319.

In one embodiment, the lipid nanoparticle formulations described hereincomprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-KC2-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about38.5% of the structural lipid cholesterol. As a non-limiting example,the lipid nanoparticle comprise about 50% of the cationic lipidDLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% ofthe PEG lipid PEG-DOMG and about 38.5% of the structural lipidcholesterol. As a non-limiting example, the lipid nanoparticle compriseabout 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

In one embodiment, the cationic lipid is selected from, but not limitedto, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent PublicationNo. US20100036115, US20120202871, US20130064894, US20130129785,US20130150625, US20130178541 and US20130225836. In another embodiment,the cationic lipid can be selected from, but not limited to, formula Adescribed in International Publication Nos. WO2012040184, WO2011153120,WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259,WO2012054365, WO2012044638 and WO2013116126 or US Patent Publication No.US20130178541 and US20130225836. In yet another embodiment, the cationiclipid can be selected from, but not limited to, formula CLI-CLXXIX ofInternational Publication No. WO2008103276, formula CLI-CLXXIX of U.S.Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 andformula I-VI of US Patent Publication No. US20100036115, formula I of USPatent Publication No US20130123338. As a non-limiting example, thecationic lipid can be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid is a cleavable lipid such as thosedescribed in International Publication No. WO2012170889. In anotherembodiment, the lipid is a cationic lipid such as, but not limited to,Formula (I) of U.S. Patent Application No. US20130064894.

In one embodiment, the cationic lipid is synthesized by methods known inthe art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2013086373 and WO2013086354.

In another embodiment, the cationic lipid is a trialkyl cationic lipid.Non-limiting examples of trialkyl cationic lipids and methods of makingand using the trialkyl cationic lipids are described in InternationalPatent Publication No. WO2013126803.

In one embodiment, the LNP formulations of the polynucleotides containPEG-c-DOMG at 3% lipid molar ratio. In another embodiment, the LNPformulations of the polynucleotides contain PEG-c-DOMG at 1.5% lipidmolar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides comprising an mRNA encoding an OX40L polypeptide includeat least one of the PEGylated lipids described in InternationalPublication No. WO2012099755.

In one embodiment, the LNP formulation contains PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation can containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation contains PEG-DMG2000, a cationic lipid known in the art, DSPC and cholesterol. As anon-limiting example, the LNP formulation contains PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation contains PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in amolar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery ofself-amplifying RNA vaccines, PNAS 2012; PMID: 22908294).

In one embodiment, the LNP formulation is formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276. As a non-limiting example, the polynucleotides comprisingan mRNA encoding an OX40L polypeptide described herein are encapsulatedin LNP formulations as described in WO2011127255 and/or WO2008103276.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide described herein are formulated in a nanoparticle tobe delivered by a parenteral route as described in U.S. Pub. No.US20120207845.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a lipid nanoparticle made by themethods described in US Patent Publication No US20130156845 orInternational Publication No WO2013093648 or WO2012024526.

The lipid nanoparticles described herein can be made in a sterileenvironment by the system and/or methods described in US PatentPublication No. US20130164400.

In one embodiment, the LNP formulation is formulated in a nanoparticlesuch as a nucleic acid-lipid particle described in U.S. Pat. No.8,492,359. As a non-limiting example, the lipid particle comprises oneor more active agents or therapeutic agents; one or more cationic lipidscomprising from about 50 mol % to about 85 mol % of the total lipidpresent in the particle; one or more non-cationic lipids comprising fromabout 13 mol % to about 49.5 mol % of the total lipid present in theparticle; and one or more conjugated lipids that inhibit aggregation ofparticles comprising from about 0.5 mol % to about 2 mol % of the totallipid present in the particle. The nucleic acid in the nanoparticle canbe the polynucleotides described herein and/or are known in the art.

In one embodiment, the LNP formulation is formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276. As a non-limiting example, modified RNA described hereinis encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276.

In one embodiment, LNP formulations described herein comprise apolycationic composition. As a non-limiting example, the polycationiccomposition is selected from formula 1-60 of US Patent Publication No.US20050222064. In another embodiment, the LNP formulations comprising apolycationic composition are used for the delivery of the modified RNAdescribed herein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein additionallycomprise a permeability enhancer molecule. Non-limiting permeabilityenhancer molecules are described in US Patent Publication No.US20050222064.

In one embodiment, the polynucleotide pharmaceutical compositions areformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes(Quiet Therapeutics, Israel).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a lyophilized gel-phase liposomalcomposition as described in US Publication No. US2012060293.

The nanoparticle formulations can comprise a phosphate conjugate. Thephosphate conjugate can increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present disclosure can be made by the methods describedin International Application No. WO2013033438 or US Patent PublicationNo. US20130196948. As a non-limiting example, the phosphate conjugatescan include a compound of any one of the formulas described inInternational Application No. WO2013033438.

The nanoparticle formulation can comprise a polymer conjugate. Thepolymer conjugate can be a water soluble conjugate. The polymerconjugate can have a structure as described in U.S. Patent ApplicationNo. 20130059360. In one aspect, polymer conjugates with thepolynucleotides comprising an mRNA encoding an OX40L polypeptide of thepresent disclosure can be made using the methods and/or segmentedpolymeric reagents described in U.S. Patent Application No. 20130072709.In another aspect, the polymer conjugate can have pendant side groupscomprising ring moieties such as, but not limited to, the polymerconjugates described in US Patent Publication No. US20130196948.

The nanoparticle formulations can comprise a conjugate to enhance thedelivery of nanoparticles of the present disclosure in a subject.Further, the conjugate can inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate is a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al (Science 2013 339, 971-975)). Asshown by Rodriguez et al. the self peptides delayed macrophage-mediatedclearance of nanoparticles which enhanced delivery of the nanoparticles.In another aspect, the conjugate is the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975). Rodriguez et al. showedthat, similarly to “self” peptides, CD47 can increase the circulatingparticle ratio in a subject as compared to scrambled peptides and PEGcoated nanoparticles.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide of the present disclosure are formulated innanoparticles which comprise a conjugate to enhance the delivery of thenanoparticles of the present disclosure in a subject. The conjugate canbe the CD47 membrane or the conjugate can be derived from the CD47membrane protein, such as the “self” peptide described previously. Inanother aspect the nanoparticle can comprise PEG and a conjugate of CD47or a derivative thereof. In yet another aspect, the nanoparticlecomprises both the “self” peptide described above and the membraneprotein CD47.

In another aspect, a “self” peptide and/or CD47 protein is conjugated toa virus-like particle or pseudovirion, as described herein for deliveryof the polynucleotides of the present disclosure.

In another embodiment, pharmaceutical compositions comprising thepolynucleotides comprising an mRNA encoding an OX40L polypeptide of thepresent disclosure and a conjugate which can have a degradable linkage.Non-limiting examples of conjugates include an aromatic moietycomprising an ionizable hydrogen atom, a spacer moiety, and awater-soluble polymer. As a non-limiting example, pharmaceuticalcompositions comprising a conjugate with a degradable linkage andmethods for delivering such pharmaceutical compositions are described inUS Patent Publication No. US20130184443.

The nanoparticle formulations can be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a polynucleotide. As anon-limiting example, the carbohydrate carrier includes, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012109121).

Nanoparticle formulations of the present disclosure can be coated with asurfactant or polymer in order to improve the delivery of the particle.In one embodiment, the nanoparticle is coated with a hydrophilic coatingsuch as, but not limited to, PEG coatings and/or coatings that have aneutral surface charge. The hydrophilic coatings can help to delivernanoparticles with larger payloads such as, but not limited to,polynucleotides within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in US Patent Publication No.US20130183244.

In one embodiment, the lipid nanoparticles of the present disclosure arehydrophilic polymer particles. Non-limiting examples of hydrophilicpolymer particles and methods of making hydrophilic polymer particlesare described in US Patent Publication No. US20130210991.

In another embodiment, the lipid nanoparticles of the present disclosureare hydrophobic polymer particles. Lipid nanoparticle formulations canbe improved by replacing the cationic lipid with a biodegradablecationic lipid which is known as a rapidly eliminated lipid nanoparticle(reLNP). Ionizable cationic lipids, such as, but not limited to,DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulatein plasma and tissues over time and can be a potential source oftoxicity. The rapid metabolism of the rapidly eliminated lipids canimprove the tolerability and therapeutic index of the lipidnanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kgdose in rat. Inclusion of an enzymatically degraded ester linkage canimprove the degradation and metabolism profile of the cationiccomponent, while still maintaining the activity of the reLNPformulation. The ester linkage can be internally located within thelipid chain or it can be terminally located at the terminal end of thelipid chain. The internal ester linkage can replace any carbon in thelipid chain.

In one embodiment, the internal ester linkage is located on either sideof the saturated carbon.

In one embodiment, an immune response is elicited by delivering a lipidnanoparticle which can include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805). The polymer can encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen canbe a recombinant protein, a modified RNA and/or an OX40L polynucleotidedescribed herein.

Lipid nanoparticles can be engineered to alter the surface properties ofparticles so the lipid nanoparticles can penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles can be removed from the mucosal tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171). The transport of nanoparticles can be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier can be madeas described in U.S. Pat. No. 8,241,670 or International PatentPublication No. WO2013110028.

The lipid nanoparticle engineered to penetrate mucus can comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material caninclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material can bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Patent PublicationNo. WO2013116804. The polymeric material can additionally be irradiated.As a non-limiting example, the polymeric material can be gammairradiated (See e.g., International App. No. WO201282165). Non-limitingexamples of specific polymers include poly(caprolactone) (PCL), ethylenevinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lacticacid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolicacid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA),poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers andmixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle can be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476), and (poly(ethyleneglycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblockcopolymer (see e.g., US Publication 20120121718 and US Publication20100003337 and U.S. Pat. No. 8,263,665). The co-polymer can be apolymer that is generally regarded as safe (GRAS) and the formation ofthe lipid nanoparticle can be in such a way that no new chemicalentities are created. For example, the lipid nanoparticle can comprisepoloxamers coating PLGA nanoparticles without forming new chemicalentities which are still able to rapidly penetrate human mucus (Yang etal. Angew. Chem. Int. Ed. 2011 50:2597-2600). A non-limiting scalablemethod to produce nanoparticles which can penetrate human mucus isdescribed by Xu et al. (See e.g., J Control Release 2013,170(2):279-86).

The vitamin of the polymer-vitamin conjugate can be vitamin E. Thevitamin portion of the conjugate can be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus can include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecyl-ammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin 34 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase. The surface altering agent can be embedded orenmeshed in the particle's surface or disposed (e.g., by coating,adsorption, covalent linkage, or other process) on the surface of thelipid nanoparticle. (see e.g., US Publication 20100215580 and USPublication 20080166414 and US20130164343).

In one embodiment, the mucus penetrating lipid nanoparticles comprisesat least one polynucleotide described herein. The polynucleotide can beencapsulated in the lipid nanoparticle and/or disposed on the surface ofthe particle. The polynucleotide can be covalently coupled to the lipidnanoparticle. Formulations of mucus penetrating lipid nanoparticles cancomprise a plurality of nanoparticles. Further, the formulations cancontain particles which can interact with the mucus and alter thestructural and/or adhesive properties of the surrounding mucus todecrease mucoadhesion which can increase the delivery of the mucuspenetrating lipid nanoparticles to the mucosal tissue.

In another embodiment, the mucus penetrating lipid nanoparticles are ahypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation can be hypotonic for the epithelium to which itis being delivered. Non-limiting examples of hypotonic formulations canbe found in International Patent Publication No. WO2013110028.

In one embodiment, in order to enhance the delivery through the mucosalbarrier the polynucleotide formulation comprises or is a hypotonicsolution. Hypotonic solutions were found to increase the rate at whichmucoinert particles such as, but not limited to, mucus-penetratingparticles, were able to reach the vaginal epithelial surface (See e.g.,Ensign et al. Biomaterials 2013 34(28):6922-9).

In one embodiment, the polynucleotide is formulated as a lipoplex, suchas, without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798;Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al.,Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al.Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 200932:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo ExpertOpin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother.34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al.,Proc Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum GeneTher. 2008 19:125-132).

In one embodiment such formulations are also constructed or compositionsaltered such that they passively or actively are directed to differentcell types in vivo, including but not limited to hepatocytes, immunecells, tumor cells, endothelial cells, antigen presenting cells, andleukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., NatBiotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel etal., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 200613:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344;Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, ExpertOpin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133). One example ofpassive targeting of formulations to liver cells includes the DLin-DMA,DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticle formulationswhich have been shown to bind to apolipoprotein E and promote bindingand uptake of these formulations into hepatocytes in vivo (Akinc et al.Mol Ther. 2010 18:1357-1364). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide of the disclosure are formulated as a solid lipidnanoparticle. A solid lipid nanoparticle (SLN) can be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and can be stabilizedwith surfactants and/or emulsifiers. In a further embodiment, the lipidnanoparticle can be a self-assembly lipid-polymer nanoparticle (seeZhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702). As a non-limitingexample, the SLN can be the SLN described in International PatentPublication No. WO2013105101. As another non-limiting example, the SLNcan be made by the methods or processes described in InternationalPatent Publication No. WO2013105101.

Liposomes, lipoplexes, or lipid nanoparticles can be used to improve theefficacy of the polynucleotides comprising an mRNA encoding an OX40Lpolypeptide as these formulations can be able to increase celltransfection by the polynucleotides; and/or increase the translation ofencoded OX40L. One such example involves the use of lipid encapsulationto enable the effective systemic delivery of polyplex plasmid DNA (Heyeset al., Mol Ther. 2007 15:713-720). The liposomes, lipoplexes, or lipidnanoparticles can also be used to increase the stability of thepolynucleotide.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide of the present disclosure are formulated forcontrolled release and/or targeted delivery. As used herein, “controlledrelease” refers to a pharmaceutical composition or compound releaseprofile that conforms to a particular pattern of release to effect atherapeutic outcome. In one embodiment, the polynucleotides areencapsulated into a delivery agent described herein and/or known in theart for controlled release and/or targeted delivery. As used herein, theterm “encapsulate” means to enclose, surround or encase. As it relatesto the formulation of the compounds of the disclosure, encapsulation canbe substantial, complete or partial. The term “substantiallyencapsulated” means that at least greater than 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of thepharmaceutical composition or compound of the disclosure can beenclosed, surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the disclosure can beenclosed, surrounded or encased within the delivery agent.Advantageously, encapsulation can be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of thedisclosure using fluorescence and/or electron micrograph. For example,at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98,99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical compositionor compound of the disclosure are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation includes, but isnot limited to, tri-block co-polymers. As a non-limiting example, theformulation includes two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106).

In another embodiment, the polynucleotides comprising an mRNA encodingan OX40L polypeptide is encapsulated into a lipid nanoparticle or arapidly eliminated lipid nanoparticle and the lipid nanoparticles or arapidly eliminated lipid nanoparticle can then be encapsulated into apolymer, hydrogel and/or surgical sealant described herein and/or knownin the art. As a non-limiting example, the polymer, hydrogel or surgicalsealant is PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics,San Diego Calif.), surgical sealants such as fibrinogen polymers(Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, IncDeerfield, Ill.), PEG-based sealants, or COSEAL® (Baxter International,Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle is encapsulated into anypolymer known in the art which can form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle isencapsulated into a polymer matrix which can be biodegradable.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide formulation for controlled release and/or targeteddelivery also includes at least one controlled release coating.Controlled release coatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the polynucleotide controlled release and/or targeteddelivery formulation comprise at least one degradable polyester whichcan contain polycationic side chains. Degradeable polyesters include,but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters can include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polynucleotide controlled release and/or targeteddelivery formulation comprising at least one polynucleotide comprises atleast one PEG and/or PEG related polymer derivatives as described inU.S. Pat. No. 8,404,222.

In another embodiment, the polynucleotide controlled release deliveryformulation comprising at least one polynucleotide is the controlledrelease polymer system described in US20130130348.

In one embodiment, the polynucleotide comprising an mRNA encoding anOX40L polypeptide of the present disclosure is encapsulated in atherapeutic nanoparticle. Therapeutic nanoparticles can be formulated bymethods described herein and known in the art such as, but not limitedto, International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286,US20120288541, US20130123351 and US20130230567 and U.S. Pat. Nos.8,206,747, 8,293,276, 8,318,208 and 8,318,211. In another embodiment,therapeutic polymer nanoparticles can be identified by the methodsdescribed in US Pub No. US20120140790.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated for sustained release. As used herein,“sustained release” refers to a pharmaceutical composition or compoundthat conforms to a release rate over a specific period of time. Theperiod of time can include, but is not limited to, hours, days, weeks,months and years. As a non-limiting example, the sustained releasenanoparticle comprises a polymer and a therapeutic agent such as, butnot limited to, the polynucleotides comprising an mRNA encoding an OX40Lpolypeptide of the present disclosure (see International Pub No.2010075072 and US Pub No. US20100216804, US20110217377 andUS20120201859). In another non-limiting example, the sustained releaseformulation comprises agents which permit persistent bioavailabilitysuch as, but not limited to, crystals, macromolecular gels and/orparticulate suspensions (see US Patent Publication No US20130150295).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide can be formulated to be target specific. Asnon-limiting examples, the therapeutic nanoparticles include acorticosteroid (see International Pub. No. WO2011084518). As anon-limiting example, the therapeutic nanoparticles are formulated innanoparticles described in International Pub No. WO2008121949,WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426,US20120004293 and US20100104655.

In one embodiment, the nanoparticles of the present disclosure comprisea polymeric matrix. As a non-limiting example, the nanoparticlecomprises two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer includes PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In yet anotherembodiment, the diblock copolymer is a high-X diblock copolymer such asthose described in International Patent Publication No. WO2013120052.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 andInternational Publication No. WO2012166923). In yet another non-limitingexample, the therapeutic nanoparticle is a stealth nanoparticle or atarget-specific stealth nanoparticle as described in US PatentPublication No. US20130172406.

In one embodiment, the therapeutic nanoparticle comprises a multiblockcopolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and USPatent Pub. No. US20130195987).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee etal. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000;as a controlled gene delivery system in Li et al. Controlled GeneDelivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253). The polynucleotides comprising an mRNA encoding an OX40Lpolypeptide of the present disclosure can be formulated in lipidnanoparticles comprising the PEG-PLGA-PEG block copolymer.

In one embodiment, the therapeutic nanoparticle comprises a multiblockcopolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and USPatent Pub. No. US20130195987).

In one embodiment, the block copolymers described herein are included ina polyion complex comprising a non-polymeric micelle and the blockcopolymer. (See e.g., U.S. Pub. No. 20120076836).

In one embodiment, the therapeutic nanoparticle comprises at least oneacrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles comprise at least onepoly(vinyl ester) polymer. The poly(vinyl ester) polymer can be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer has a structure such as those described inInternational Application No. WO2013032829 or US Patent Publication NoUS20130121954. In one aspect, the poly(vinyl ester) polymers can beconjugated to the polynucleotides described herein.

In one embodiment, the therapeutic nanoparticle comprises at least onediblock copolymer. The diblock copolymer can be, but is not limited to,a poly(lactic) acid-poly(ethylene)glycol copolymer (see e.g.,International Patent Publication No. WO2013044219). As a non-limitingexample, the therapeutic nanoparticle are used to treat cancer (seeInternational publication No. WO2013044219).

In one embodiment, the therapeutic nanoparticles comprise at least onecationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles comprise at least oneamine-containing polymer such as, but are not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(See e.g., U.S. Pat. No. 8,287,849) and combinations thereof.

In another embodiment, the nanoparticles described herein comprise anamine cationic lipid such as those described in International PatentApplication No. WO2013059496. In one aspect the cationic lipids have anamino-amine or an amino-amide moiety.

In one embodiment, the therapeutic nanoparticles comprise at least onedegradable polyester which can contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters can include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle includes aconjugation of at least one targeting ligand. The targeting ligand canbe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740).

In one embodiment, the therapeutic nanoparticle is formulated in anaqueous solution which can be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide is formulated using the methods described byPodobinski et al in U.S. Pat. No. 8,404,799.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide is encapsulated in, linked to and/or associated withsynthetic nanocarriers. Synthetic nanocarriers include, but are notlimited to, those described in International Pub. Nos. WO2010005740,WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259,WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393,WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US Pub.Nos. US20110262491, US20100104645, US20100087337 and US20120244222. Thesynthetic nanocarriers can be formulated using methods known in the artand/or described herein. As a non-limiting example, the syntheticnanocarriers can be formulated by the methods described in InternationalPub Nos. WO2010005740, WO2010030763 and WO201213501 and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US2012024422. In anotherembodiment, the synthetic nanocarrier formulations can be lyophilized bymethods described in International Pub. No. WO2011072218 and U.S. Pat.No. 8,211,473. In yet another embodiment, formulations of the presentdisclosure, including, but not limited to, synthetic nanocarriers, canbe lyophilized or reconstituted by the methods described in US PatentPublication No. US20130230568.

In one embodiment, the synthetic nanocarriers contain reactive groups torelease the polynucleotides described herein (see International Pub. No.WO20120952552 and US Pub No. US20120171229).

In one embodiment, the synthetic nanocarriers contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier can comprise a Th1 immunostimulatory agent which can enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201).

In one embodiment, the synthetic nanocarriers are formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle is formulated to release the polynucleotides after 24 hoursand/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 andWO2010138194 and US Pub Nos. US20110020388 and US20110027217).

In one embodiment, the synthetic nanocarriers are formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release are formulated by methods known in the art, describedherein and/or as described in International Pub No. WO2010138192 and USPub No. 20100303850.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated for controlled and/or sustained releasewherein the formulation comprises at least one polymer that is acrystalline side chain (CYSC) polymer. CYSC polymers are described inU.S. Pat. No. 8,399,007.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are encapsulated in, linked to and/or associated withzwitterionic lipids. Non-limiting examples of zwitterionic lipids andmethods of using zwitterionic lipids are described in US PatentPublication No. US20130216607. In one aspect, the zwitterionic lipidscan be used in the liposomes and lipid nanoparticles described herein.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in colloid nanocarriers as described inUS Patent Publication No. US20130197100.

In one embodiment, the nanoparticle is optimized for oraladministration. The nanoparticle can comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle can be formulatedby the methods described in U.S. Pub. No. 20120282343.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832). Activityand/or safety (as measured by examining one or more of ALT/AST, whiteblood cell count and cytokine induction) of LNP administration can beimproved by incorporation of such lipids. LNPs comprising KL52 can beadministered intravenously and/or in one or more doses. In someembodiments, administration of LNPs comprising KL52 results in equal orimproved mRNA and/or protein expression as compared to LNPs comprisingMC3.

In some embodiments, polynucleotides comprising an mRNA encoding anOX40L polypeptide are delivered using smaller LNPs. Such particles cancomprise a diameter from below 0.1 um up to 100 nm such as, but notlimited to, less than 0.1 um, less than 1.0 um, less than 5 um, lessthan 10 um, less than 15 um, less than 20 um, less than 25 um, less than30 um, less than 35 um, less than 40 um, less than 50 um, less than 55um, less than 60 um, less than 65 um, less than 70 um, less than 75 um,less than 80 um, less than 85 um, less than 90 um, less than 95 um, lessthan 100 um, less than 125 um, less than 150 um, less than 175 um, lessthan 200 um, less than 225 um, less than 250 um, less than 275 um, lessthan 300 um, less than 325 um, less than 350 um, less than 375 um, lessthan 400 um, less than 425 um, less than 450 um, less than 475 um, lessthan 500 um, less than 525 um, less than 550 um, less than 575 um, lessthan 600 um, less than 625 um, less than 650 um, less than 675 um, lessthan 700 um, less than 725 um, less than 750 um, less than 775 um, lessthan 800 um, less than 825 um, less than 850 um, less than 875 um, lessthan 900 um, less than 925 um, less than 950 um, or less than 975 um.

In another embodiment, polynucleotides comprising an mRNA encoding anOX40L polypeptide are delivered using smaller LNPs which can comprise adiameter from about 1 nm to about 100 nm, from about 1 nm to about 10nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, fromabout 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm toabout 80 nm, from about 1 nm to about 90 nm, from about 5 nm to aboutfrom 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm,from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, fromabout 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm toabout 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, fromabout 30 to about 50 nm, from about 40 to about 50 nm, from about 20 toabout 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm,from about 20 to about 70 nm, from about 30 to about 70 nm, from about40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, fromabout 40 to about 80 nm, from about 50 to about 80 nm, from about 60 toabout 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm,from about 40 to about 90 nm, from about 50 to about 90 nm, from about60 to about 90 nm and/or from about 70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Exemplary microfluidic mixers can include, but arenot limited to a slit interdigitial micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51). In some embodiments, methods of LNP generationcomprising SHM, further comprise the mixing of at least two inputstreams wherein mixing occurs by microstructure-induced chaoticadvection (MICA). According to this method, fluid streams flow throughchannels present in a herringbone pattern causing rotational flow andfolding the fluids around each other. This method can also comprise asurface for fluid mixing wherein the surface changes orientations duringfluid cycling. Methods of generating LNPs using SHM include thosedisclosed in U.S. Application Publication Nos. 2004/0262223 and2012/0276209.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide of the present disclosure is formulated in lipidnanoparticles created using a micromixer such as, but not limited to, aSlit Interdigital Microstructured Mixer (SIMM-V2) or a Standard SlitInterdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet(IJMM) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide of the present disclosure are formulated in lipidnanoparticles created using microfluidic technology (see Whitesides,George M. The Origins and the Future of Microfluidics. Nature, 2006 442:368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science,2002 295: 647-651). As a non-limiting example, controlled microfluidicformulation includes a passive method for mixing streams of steadypressure-driven flows in micro channels at a low Reynolds number (Seee.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide can be formulated in lipid nanoparticles created usinga micromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated for delivery using the drugencapsulating microspheres described in International Patent PublicationNo. WO2013063468 or U.S. Pat. No. 8,440,614. The microspheres cancomprise a compound of the formula (I), (II), (III), (IV), (V) or (VI)as described in International Patent Publication No. WO2013063468. Inanother aspect, the amino acid, peptide, polypeptide, lipids (APPL) areuseful in delivering the polynucleotides of the disclosure to cells (seeInternational Patent Publication No. WO2013063468).

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in lipid nanoparticles having adiameter from about 10 to about 100 nm such as, but not limited to,about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 toabout 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm,about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 toabout 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm,about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 toabout 100 nm and/or about 90 to about 100 nm.

In one embodiment, the lipid nanoparticles have a diameter from about 10to 500 nm. In one embodiment, the lipid nanoparticle has a diametergreater than 100 nm, greater than 150 nm, greater than 200 nm, greaterthan 250 nm, greater than 300 nm, greater than 350 nm, greater than 400nm, greater than 450 nm, greater than 500 nm, greater than 550 nm,greater than 600 nm, greater than 650 nm, greater than 700 nm, greaterthan 750 nm, greater than 800 nm, greater than 850 nm, greater than 900nm, greater than 950 nm or greater than 1000 nm.

In one aspect, the lipid nanoparticle is a limit size lipid nanoparticledescribed in International Patent Publication No. WO2013059922. Thelimit size lipid nanoparticle can comprise a lipid bilayer surroundingan aqueous core or a hydrophobic core; where the lipid bilayer cancomprise a phospholipid such as, but not limited to,diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide,a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, aC8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect the limit size lipidnanoparticle can comprise a polyethylene glycol-lipid such as, but notlimited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide is delivered, localized and/or concentrated in aspecific location using the delivery methods described in InternationalPatent Publication No. WO2013063530. As a non-limiting example, asubject can be administered an empty polymeric particle prior to,simultaneously with or after delivering the polynucleotides to thesubject. The empty polymeric particle undergoes a change in volume oncein contact with the subject and becomes lodged, embedded, immobilized orentrapped at a specific location in the subject.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in an active substance release system(See e.g., US Patent Publication No. US20130102545). The activesubstance release system can comprise 1) at least one nanoparticlebonded to an oligonucleotide inhibitor strand which is hybridized with acatalytically active nucleic acid and 2) a compound bonded to at leastone substrate molecule bonded to a therapeutically active substance(e.g., polynucleotides described herein), where the therapeuticallyactive substance is released by the cleavage of the substrate moleculeby the catalytically active nucleic acid.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a nanoparticle comprising an innercore comprising a non-cellular material and an outer surface comprisinga cellular membrane. The cellular membrane can be derived from a cell ora membrane derived from a virus. As a non-limiting example, thenanoparticle is made by the methods described in International PatentPublication No. WO2013052167. As another non-limiting example, thenanoparticle described in International Patent Publication No.WO2013052167, is used to deliver the polynucleotides described herein.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in porous nanoparticle-supported lipidbilayers (protocells). Protocells are described in International PatentPublication No. WO2013056132.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide described herein are formulated in polymericnanoparticles as described in or made by the methods described in U.S.Pat. Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073 848B1.As a non-limiting example, the polymeric nanoparticle has a high glasstransition temperature such as the nanoparticles described in ornanoparticles made by the methods described in U.S. Pat. No. 8,518,963.As another non-limiting example, the polymer nanoparticle for oral andparenteral formulations is made by the methods described in EuropeanPatent No. EP2073848B1.

In another embodiment, the polynucleotides comprising an mRNA encodingan OX40L polypeptide described herein are formulated in nanoparticlesused in imaging. The nanoparticles can be liposome nanoparticles such asthose described in US Patent Publication No US20130129636. As anon-limiting example, the liposome can comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see e.g., USPatent Publication No US20130129636).

In one embodiment, the nanoparticles which can be used in the presentdisclosure are formed by the methods described in U.S. PatentApplication No. US20130130348.

The nanoparticles of the present disclosure can further includenutrients such as, but not limited to, those which deficiencies can leadto health hazards from anemia to neural tube defects (see e.g, thenanoparticles described in International Patent Publication NoWO2013072929). As a non-limiting example, the nutrient is iron in theform of ferrous, ferric salts or elemental iron, iodine, folic acid,vitamins or micronutrients.

In one embodiment, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a swellable nanoparticle. Theswellable nanoparticle can be, but is not limited to, those described inU.S. Pat. No. 8,440,231. As a non-limiting embodiment, the swellablenanoparticle is used for delivery of the polynucleotides of the presentdisclosure to the pulmonary system (see e.g., U.S. Pat. No. 8,440,231).

The polynucleotides comprising an mRNA encoding an OX40L polypeptide areformulated in polyanhydride nanoparticles such as, but not limited to,those described in U.S. Pat. No. 8,449,916.

The nanoparticles and microparticles of the present disclosure can begeometrically engineered to modulate macrophage and/or the immuneresponse. In one aspect, the geometrically engineered particles can havevaried shapes, sizes and/or surface charges in order to incorporated thepolynucleotides of the present disclosure for targeted delivery such as,but not limited to, pulmonary delivery (see e.g., InternationalPublication No WO2013082111). Other physical features the geometricallyengineering particles can have include, but are not limited to,fenestrations, angled arms, asymmetry and surface roughness, chargewhich can alter the interactions with cells and tissues. As anon-limiting example, nanoparticles of the present disclosure are madeby the methods described in International Publication No WO2013082111.

In one embodiment, the nanoparticles of the present disclosure are watersoluble nanoparticles such as, but not limited to, those described inInternational Publication No. WO2013090601. The nanoparticles can beinorganic nanoparticles which have a compact and zwitterionic ligand inorder to exhibit good water solubility. The nanoparticles can also havesmall hydrodynamic diameters (HD), stability with respect to time, pH,and salinity and a low level of non-specific protein binding.

In one embodiment the nanoparticles of the present disclosure aredeveloped by the methods described in US Patent Publication No.US20130172406.

In one embodiment, the nanoparticles of the present disclosure arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in US Patent Publication No.US20130172406. The nanoparticles of the present disclosure can be madeby the methods described in US Patent Publication No. US20130172406.

In another embodiment, the stealth or target-specific stealthnanoparticles comprise a polymeric matrix. The polymeric matrix cancomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In one embodiment, the nanoparticle is a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure ismade by the methods described in US Patent Publication No.US20130171646. The nanoparticle can comprise a nucleic acid such as, butnot limited to, polynucleotides described herein and/or known in theart.

At least one of the nanoparticles of the present disclosure can beembedded in the core a nanostructure or coated with a low density porous3-D structure or coating which is capable of carrying or associatingwith at least one payload within or on the surface of the nanostructure.Non-limiting examples of the nanostructures comprising at least onenanoparticle are described in International Patent Publication No.WO2013123523.

Hyaluronidase

The intramuscular, intratumoral, or subcutaneous localized injection ofpolynucleotides comprising an mRNA encoding an OX40L polypeptide caninclude hyaluronidase, which catalyzes the hydrolysis of hyaluronan. Bycatalyzing the hydrolysis of hyaluronan, a constituent of theinterstitial barrier, hyaluronidase lowers the viscosity of hyaluronan,thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv.(2007) 4:427-440). It is useful to speed their dispersion and systemicdistribution of encoded proteins produced by transfected cells.Alternatively, the hyaluronidase can be used to increase the number ofcells exposed to a polynucleotide of the disclosure administeredintramuscularly, intratumorally, or subcutaneously.

Nanoparticle Mimics

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe encapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotides of the disclosure can be encapsulated in a non-virionparticle which can mimic the delivery function of a virus (seeInternational Pub. No. WO2012006376 and US Patent Publication No.US20130171241 and US20130195968).

Nanotubes

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe attached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Thepolynucleotides can be bound to the nanotubes through forces such as,but not limited to, steric, ionic, covalent and/or other forces.Nanotubes and nanotube formulations comprising polynucleotides aredescribed in International Patent Application No. PCT/US2014/027077.

Self-Assembled Nanoparticles

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in self-assembled nanoparticles. Nucleic acidself-assembled nanoparticles are described in International PatentApplication No. PCT/US2014/027077, such as in paragraphs[000740]-[000743]. Polymer-based self-assembled nanoparticles aredescribed in International Patent Application No. PCT/US2014/027077.

Self-Assembled Macromolecules

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in amphiphilic macromolecules (AMs) for delivery. AMscomprise biocompatible amphiphilic polymers which have an alkylatedsugar backbone covalently linked to poly(ethylene glycol). In aqueoussolution, the AMs self-assemble to form micelles. Non-limiting examplesof methods of forming AMs and AMs are described in US Patent PublicationNo. US20130217753.

Inorganic Nanoparticles

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in inorganic nanoparticles (U.S. Pat. No. 8,257,745). Theinorganic nanoparticles can include, but are not limited to, claysubstances that are water swellable. As a non-limiting example, theinorganic nanoparticle include synthetic smectite clays which are madefrom simple silicates (See e.g., U.S. Pat. Nos. 5,585,108 and8,257,745).

In some embodiments, the inorganic nanoparticles comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellcan be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell can be used to protect thepolynucleotides in the core.

Semi-Conductive and Metallic Nanoparticles

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in water-dispersible nanoparticle comprising asemiconductive or metallic material (U.S. Pub. No. 20120228565) orformed in a magnetic nanoparticle (U.S. Pub. No. 20120265001 and20120283503). The water-dispersible nanoparticles can be hydrophobicnanoparticles or hydrophilic nanoparticles.

In some embodiments, the semi-conductive and/or metallic nanoparticlescan comprise a core of the polynucleotides disclosed herein and apolymer shell. The polymer shell can be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell can be used to protect the polynucleotides in the core.

Surgical Sealants: Gels and Hydrogels

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are encapsulated into any hydrogel known in the artwhich forms a gel when injected into a subject. Surgical sealants suchas gels and hydrogels are described in International Patent ApplicationNo. PCT/US2014/027077.

Suspension Formulations

In some embodiments, suspension formulations are provided comprisingpolynucleotides, water immiscible oil depots, surfactants and/orco-surfactants and/or co-solvents. Combinations of oils and surfactantscan enable suspension formulation with polynucleotides. Delivery ofpolynucleotides in a water immiscible depot can be used to improvebioavailability through sustained release of mRNA from the depot to thesurrounding physiologic environment and prevent polynucleotidesdegradation by nucleases.

In some embodiments, suspension formulations of mRNA are prepared usingcombinations of polynucleotides, oil-based solutions and surfactants.Such formulations can be prepared as a two-part system comprising anaqueous phase comprising polynucleotides and an oil-based phasecomprising oil and surfactants. Exemplary oils for suspensionformulations can include, but are not limited to sesame oil and Miglyol(comprising esters of saturated coconut and palmkernel oil-derivedcaprylic and capric fatty acids and glycerin or propylene glycol), cornoil, soybean oil, peanut oil, beeswax and/or palm seed oil. Exemplarysurfactants can include, but are not limited to Cremophor, polysorbate20, polysorbate 80, polyethylene glycol, transcutol, CAPMUL®, labrasol,isopropyl myristate, and/or Span 80. In some embodiments, suspensionscan comprise co-solvents including, but not limited to ethanol, glyceroland/or propylene glycol.

Suspensions can be formed by first preparing polynucleotides formulationcomprising an aqueous solution of polynucleotide and an oil-based phasecomprising one or more surfactants. Suspension formation occurs as aresult of mixing the two phases (aqueous and oil-based). In someembodiments, such a suspension can be delivered to an aqueous phase toform an oil-in-water emulsion. In some embodiments, delivery of asuspension to an aqueous phase results in the formation of anoil-in-water emulsion in which the oil-based phase comprisingpolynucleotides forms droplets that can range in size fromnanometer-sized droplets to micrometer-sized droplets. In someembodiments, specific combinations of oils, surfactants, cosurfactantsand/or co-solvents can be utilized to suspend polynucleotides in the oilphase and/or to form oil-in-water emulsions upon delivery into anaqueous environment.

In some embodiments, suspensions provide modulation of the release ofpolynucleotides into the surrounding environment. In such embodiments,polynucleotides release can be modulated by diffusion from a waterimmiscible depot followed by resolubilization into a surroundingenvironment (e.g. an aqueous environment).

In some embodiments, polynucleotides within a water immiscible depot(e.g. suspended within an oil phase) result in altered polynucleotidesstability (e.g. altered degradation by nucleases).

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated such that upon injection, an emulsionforms spontaneously (e.g. when delivered to an aqueous phase). Suchparticle formation can provide a high surface area to volume ratio forrelease of polynucleotides from an oil phase to an aqueous phase.

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a nanoemulsion such as, but notlimited to, the nanoemulsions described in U.S. Pat. No. 8,496,945. Thenanoemulsions can comprise nanoparticles described herein. As anon-limiting example, the nanoparticles can comprise a liquidhydrophobic core which can be surrounded or coated with a lipid orsurfactant layer. The lipid or surfactant layer can comprise at leastone membrane-integrating peptide and can also comprise a targetingligand (see e.g., U.S. Pat. No. 8,496,945).

Cations and Anions

Formulations of polynucleotides comprising an mRNA encoding an OX40Lpolypeptide can include cations or anions. In some embodiments, theformulations include metal cations such as, but not limited to, Zn2+,Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting example,formulations include polymers and a polynucleotides complexed with ametal cation (See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525).

In some embodiments, cationic nanoparticles comprising combinations ofdivalent and monovalent cations are formulated with polynucleotides.Such nanoparticles can form spontaneously in solution over a givenperiod (e.g. hours, days, etc). Such nanoparticles do not form in thepresence of divalent cations alone or in the presence of monovalentcations alone. The delivery of polynucleotides in cationic nanoparticlesor in one or more depot comprising cationic nanoparticles can improvepolynucleotide bioavailability by acting as a long-acting depot and/orreducing the rate of degradation by nucleases.

Molded Nanoparticles and Microparticles

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in nanoparticles and/or microparticles. As an example, thenanoparticles and/or microparticles can be made using the PRINT®technology by LIQUIDA TECHNOLOGIES® (Morrisville, N.C.) (See, e.g.,International Pub. No. WO2007024323).

In some embodiments, the nanoparticles comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellcan be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell can be used to protect thepolynucleotides in the core.

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in microparticles. The microparticlescan contain a core of the polynucleotides and a cortex of abiocompatible and/or biodegradable polymer. As a non-limiting example,the microparticles which can be used with the present disclosure can bethose described in U.S. Pat. No. 8,460,709, U.S. Patent Publication No.US20130129830 and International Patent Publication No WO2013075068. Asanother non-limiting example, the microparticles can be designed toextend the release of the polynucleotides of the present disclosure overa desired period of time (see e.g, extended release of a therapeuticprotein in U.S. Patent Publication No. US20130129830).

NanoJackets and NanoLiposomes

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe formulated in NanoJackets and NanoLiposomes by Keystone Nano (StateCollege, Pa.). NanoJackets are made of compounds that are naturallyfound in the body including calcium, phosphate and can also include asmall amount of silicates. Nanojackets can range in size from 5 to 50 nmand can be used to deliver hydrophilic and hydrophobic compounds suchas, but not limited to, polynucleotides.

NanoLiposomes are made of lipids such as, but not limited to, lipidswhich naturally occur in the body. NanoLiposomes can range in size from60-80 nm and can be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides. In one aspect,the polynucleotides comprising an mRNA encoding an OX40L polypeptide areformulated in a NanoLiposome such as, but not limited to, CeramideNanoLiposomes.

Minicells

In one aspect, the polynucleotides comprising an mRNA encoding an OX40Lpolypeptide can be formulated in bacterial minicells. As a non-limitingexample, bacterial minicells are those described in InternationalPublication No. WO2013088250 or US Patent Publication No. US20130177499.The bacterial minicells comprising therapeutic agents such aspolynucleotides described herein can be used to deliver the therapeuticagents to brain tumors.

Semi-Solid Compositions

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated with a hydrophobic matrix to form asemi-solid composition. As a non-limiting example, the semi-solidcomposition or paste-like composition is made by the methods describedin International Patent Publication No WO201307604. The semi-solidcomposition can be a sustained release formulation as described inInternational Patent Publication No WO201307604.

In another embodiment, the semi-solid composition further has amicro-porous membrane or a biodegradable polymer formed around thecomposition (see e.g., International Patent Publication No WO201307604).

The semi-solid composition using the polynucleotides comprising an mRNAencoding an OX40L polypeptide can have the characteristics of thesemi-solid mixture as described in International Patent Publication NoWO201307604 (e.g., a modulus of elasticity of at least 10⁻⁴ N·mm⁻²,and/or a viscosity of at least 100 mPa·s).

Exosomes

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in exosomes. The exosomes can be loadedwith at least one polynucleotide and delivered to cells, tissues and/ororganisms. As a non-limiting example, the polynucleotides comprising anmRNA encoding an OX40L polypeptide can be loaded in the exosomesdescribed in International Publication No. WO2013084000.

Silk-Based Delivery

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in a sustained release silk-baseddelivery system. The silk-based delivery system can be formed bycontacting a silk fibroin solution with a therapeutic agent such as, butnot limited to, the polynucleotides comprising an mRNA encoding an OX40Lpolypeptide. As a non-limiting example, the sustained release silk-baseddelivery system which can be used in the present disclosure and methodsof making such system are described in US Patent Publication No.US20130177611.

Microparticles

In some embodiments, formulations comprising polynucleotides comprisingan mRNA encoding an OX40L polypeptide comprise microparticles. Themicroparticles can comprise a polymer described herein and/or known inthe art such as, but not limited to, poly(α-hydroxy acid), a polyhydroxybutyric acid, a polycaprolactone, a polyorthoester and a polyanhydride.The microparticle can have adsorbent surfaces to adsorb biologicallyactive molecules such as polynucleotides. As a non-limiting examplemicroparticles for use with the present disclosure and methods of makingmicroparticles are described in US Patent Publication No. US2013195923and US20130195898 and U.S. Pat. Nos. 8,309,139 and 8,206,749.

In another embodiment, the formulation is a microemulsion comprisingmicroparticles and polynucleotides. As a non-limiting example,microemulsions comprising microparticles are described in US PatentPublication No. US2013195923 and US20130195898 and U.S. Pat. Nos.8,309,139 and 8,206,749.

Amino Acid Lipids

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in amino acid lipids. Amino acid lipidsare lipophilic compounds comprising an amino acid residue and one ormore lipophilic tails. Non-limiting examples of amino acid lipids andmethods of making amino acid lipids are described in U.S. Pat. No.8,501,824.

In some embodiments, the amino acid lipids have a hydrophilic portionand a lipophilic portion. The hydrophilic portion can be an amino acidresidue and a lipophilic portion can comprise at least one lipophilictail.

In some embodiments, the amino acid lipid formulations are used todeliver the polynucleotides to a subject.

In another embodiment, the amino acid lipid formulations deliver apolynucleotide in releasable form which comprises an amino acid lipidthat binds and releases the polynucleotides. As a non-limiting example,the release of the polynucleotides can be provided by an acid-labilelinker such as, but not limited to, those described in U.S. Pat. Nos.7,098,032, 6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931.

Microvesicles

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in microvesicles. Non-limiting examplesof microvesicles include those described in US Patent Publication No.US20130209544.

In some embodiments, the microvesicle is an ARRDC1-mediatedmicrovesicles (ARMMs). Non-limiting examples of ARMMs and methods ofmaking ARMMs are described in International Patent Publication No.WO2013119602.

Interpolyelectrolyte Complexes

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in an interpolyelectrolyte complex.Interpolyelectrolyte complexes are formed when charge-dynamic polymersare complexed with one or more anionic molecules. Non-limiting examplesof charge-dynamic polymers and interpolyelectrolyte complexes andmethods of making interpolyelectrolyte complexes are described in U.S.Pat. No. 8,524,368.

Crystalline Polymeric Systems

In some embodiments, the polynucleotides comprising an mRNA encoding anOX40L polypeptide are formulated in crystalline polymeric systems.Crystalline polymeric systems are polymers with crystalline moietiesand/or terminal units comprising crystalline moieties. Non-limitingexamples of polymers with crystalline moieties and/or terminal unitscomprising crystalline moieties termed “CYC polymers,” crystallinepolymer systems and methods of making such polymers and systems aredescribed in U.S. Pat. No. 8,524,259.

Excipients

Pharmaceutical formulations can additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but are notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants, flavoring agents, stabilizers, antioxidants,osmolality adjusting agents, pH adjusting agents and the like, as suitedto the particular dosage form desired. Various excipients forformulating pharmaceutical compositions and techniques for preparing thecomposition are known in the art (see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gennaro (Lippincott,Williams & Wilkins, Baltimore, Md., 2006). The use of a conventionalexcipient medium can be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisdisclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use for humansand for veterinary use. In some embodiments, an excipient can beapproved by United States Food and Drug Administration. In someembodiments, an excipient can be of pharmaceutical grade. In someembodiments, an excipient can meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients can optionally be included in pharmaceutical compositions.The composition can also include excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids(e.g., glycine); natural and synthetic gums (e.g. acacia, sodiumalginate, extract of Irish moss, panwar gum, ghatti gum, mucilage ofisapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives can include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Oxidation is a potential degradation pathway for mRNA,especially for liquid mRNA formulations. In order to prevent oxidation,antioxidants can be added to the formulation. Exemplary antioxidantsinclude, but are not limited to, alpha tocopherol, ascorbic acid,acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA,m-cresol, methionine, butylated hydroxytoluene, monothioglycerol,potassium metabisulfite, propionic acid, propyl gallate, sodiumascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/orsodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and/or sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidicpreservatives include, but are not limited to, vitamin A, vitamin C,vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid,ascorbic acid, sorbic acid, and/or phytic acid. Other preservativesinclude, but are not limited to, tocopherol, tocopherol acetate,deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

In some embodiments, the pH of polynucleotide solutions is maintainedbetween pH 5 and pH 8 to improve stability. Exemplary buffers to controlpH can include, but are not limited to sodium phosphate, sodium citrate,sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/orsodium malate. In another embodiment, the exemplary buffers listed abovecan be used with additional monovalent counterions (including, but notlimited to potassium). Divalent cations can also be used as buffercounterions; however, these are not preferred due to complex formationand/or mRNA degradation.

Exemplary buffering agents can also include, but are not limited to,citrate buffer solutions, acetate buffer solutions, phosphate buffersolutions, ammonium chloride, calcium carbonate, calcium chloride,calcium citrate, calcium glubionate, calcium gluceptate, calciumgluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginicacid, pyrogen-free water, isotonic saline, Ringer's solution, ethylalcohol, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary additives include physiologically biocompatible buffers (e.g.,trimethylamine hydrochloride), addition of chelants (such as, forexample, DTPA or DTPA-bisamide) or calcium chelate complexes (as forexample calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions ofcalcium or sodium salts (for example, calcium chloride, calciumascorbate, calcium gluconate or calcium lactate). In addition,antioxidants and suspending agents can be used.

Cryoprotectants for mRNA

In some embodiments, the polynucleotide formulations comprisecyroprotectants. As used herein, the term “cryoprotectant” refers to oneor more agent that when combined with a given substance, helps to reduceor eliminate damage to that substance that occurs upon freezing. In someembodiments, cryoprotectants are combined with polynucleotides in orderto stabilize them during freezing. Frozen storage of mRNA between −20°C. and −80° C. can be advantageous for long term (e.g. 36 months)stability of polynucleotide. In some embodiments, cryoprotectants areincluded in polynucleotide formulations to stabilize polynucleotidethrough freeze/thaw cycles and under frozen storage conditions.Cryoprotectants of the present disclosure can include, but are notlimited to sucrose, trehalose, lactose, glycerol, dextrose, raffinoseand/or mannitol. Trehalose is listed by the Food and Drug Administrationas being generally regarded as safe (GRAS) and is commonly used incommercial pharmaceutical formulations.

Bulking Agents

In some embodiments, the polynucleotide formulations comprise bulkingagents. As used herein, the term “bulking agent” refers to one or moreagents included in formulations to impart a desired consistency to theformulation and/or stabilization of formulation components. In someembodiments, bulking agents are included in lyophilized polynucleotideformulations to yield a “pharmaceutically elegant” cake, stabilizing thelyophilized polynucleotides during long term (e.g. 36 month) storage.Bulking agents of the present disclosure can include, but are notlimited to sucrose, trehalose, mannitol, glycine, lactose and/orraffinose. In some embodiments, combinations of cryoprotectants andbulking agents (for example, sucrose/glycine or trehalose/mannitol) canbe included to both stabilize polynucleotides during freezing andprovide a bulking agent for lyophilization.

Non-limiting examples of formulations and methods for formulating thepolynucleotides of the present disclosure are also provided inInternational Publication No WO2013090648 filed Dec. 14, 2012.

Naked Delivery

The polynucleotides comprising an mRNA encoding an OX40L polypeptide canbe delivered to a cell (e.g., to a tumor cell) naked. As used herein in,“naked” refers to delivering polynucleotides free from agents whichpromote transfection. For example, the polynucleotides delivered to thecell, e.g., tumor cell, can contain no modifications. The nakedpolynucleotides comprising an mRNA encoding an OX40L polypeptide can bedelivered to the tumor cell using routes of administration known in theart, e.g., intratumoral administration, and described herein.

Parenteral and Injectable Administration

Liquid dosage forms for parenteral administration, e.g. intratumoral,include, but are not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms can comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

A pharmaceutical composition for parenteral administration, e.g.,intratumoral administration, can comprise at least one inactiveingredient. Any or none of the inactive ingredients used can have beenapproved by the US Food and Drug Administration (FDA). A non-exhaustivelist of inactive ingredients for use in pharmaceutical compositions forparenteral administration includes hydrochloric acid, mannitol,nitrogen, sodium acetate, sodium chloride and sodium hydroxide.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations can be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. The sterileformulation can also comprise adjuvants such as local anesthetics,preservatives and buffering agents.

Injectable formulations, e.g., intratumoral, can be sterilized, forexample, by filtration through a bacterial-retaining filter, and/or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

Injectable formulations, e.g., intratumoral, can be for direct injectioninto a region of a tissue, organ and/or subject, e.g., tumor.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromintratumoral injection. This can be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, can depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intratumoral,intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal,or subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration (e.g., intratumoral)include, but are not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms can comprise inertdiluents commonly used in the art including, but not limited to, wateror other solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. In certain embodiments for parenteral administration,compositions can be mixed with solubilizing agents such as CREMOPHOR®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and/or combinations thereof.

Injectable

Injectable preparations (e.g., intratumoral), for example, sterileinjectable aqueous or oleaginous suspensions can be formulated accordingto the known art and can include suitable dispersing agents, wettingagents, and/or suspending agents. Sterile injectable preparations can besterile injectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat can be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it can bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotidesthen depends upon its rate of dissolution which, in turn, can dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered polynucleotides can beaccomplished by dissolving or suspending the polynucleotides in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the polynucleotides in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of polynucleotidesto polymer and the nature of the particular polymer employed, the rateof polynucleotides release can be controlled. Examples of otherbiodegradable polymers include, but are not limited to,poly(orthoesters) and poly(anhydrides). Depot injectable formulationscan be prepared by entrapping the polynucleotides in liposomes ormicroemulsions which are compatible with body tissues.

VII. KITS AND DEVICES Kits

The disclosure provides a variety of kits for conveniently and/oreffectively carrying out methods of the present disclosure. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present disclosure provides kits comprising thepolynucleotides of the disclosure. In some embodiments, the kitcomprises one or more polynucleotides.

The kits can be for protein production, comprising a polynucleotidecomprising an mRNA encoding an OX40L polypeptide. The kit can furthercomprise packaging and instructions and/or a delivery agent to form aformulation composition. The delivery agent can comprise a saline, abuffered solution, a lipidoid or any delivery agent disclosed herein.

In some embodiments, the buffer solution includes sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution includes, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No.20120258046). In a further embodiment, the buffer solution isprecipitated or it is lyophilized. The amount of each component can bevaried to enable consistent, reproducible higher concentration saline orsimple buffer formulations. The components can also be varied in orderto increase the stability of modified RNA in the buffer solution over aperiod of time and/or under a variety of conditions. In one aspect, thepresent disclosure provides kits for protein production, comprising: apolynucleotide comprising a translatable region, provided in an amounteffective to produce a desired amount of a protein encoded by thetranslatable region when introduced into a target cell; a secondpolynucleotide comprising an inhibitory nucleic acid, provided in anamount effective to substantially inhibit the innate immune response ofthe cell; and packaging and instructions.

In one aspect, the present disclosure provides kits for proteinproduction, comprising a polynucleotide comprising an mRNA encoding anOX40L polypeptide, wherein the polynucleotide exhibits reduceddegradation by a cellular nuclease, and packaging and instructions.

Devices

The present disclosure provides for devices which can incorporatepolynucleotides comprising an mRNA encoding an OX40L polypeptide. Thesedevices contain in a stable formulation the reagents to synthesize apolynucleotide in a formulation available to be immediately delivered toa subject in need thereof, such as a human patient.

Devices for administration can be employed to deliver thepolynucleotides comprising an mRNA encoding an OX40L polypeptideaccording to single, multi- or split-dosing regimens taught herein. Suchdevices are taught in, for example, International Publication No. WO2013151666 A2.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentdisclosure. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present disclosure, these multi-administration devicescan be utilized to deliver the single, multi- or split dosescontemplated herein. Such devices are taught for example in,International Publication No. WO 2013151666 A2.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art can be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they can beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the disclosure (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, patents, publications,databases, database entries, and art cited herein, are incorporated intothis application by reference, even if not expressly stated in thecitation. In case of conflicting statements of a cited source and theinstant application, the statement in the instant application shallcontrol.

Section and table headings are not intended to be limiting.

Examples Example 1. In Vitro Cell Surface Expression of an OX40LPolypeptide

Expression of an OX40L polypeptide was measured on the surface of cancercells following treatment with a polynucleotide comprising an mRNAencoding an OX40L polypeptide.

A. Formulation of mOX40L_miR122

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasused in this example (mOX40L_miR122; SEQ ID NO: 66). The OX40L modifiedmRNA was formulated in lipid nanoparticles (LNP) as described herein.(Moderna Therapeutics, Cambridge, Mass.).

B. Analysis of OX40L Cell-Surface Expression

Mouse melanoma cells (B16F10, ATCC No. CRL-6475; ATCC, Manassas, Va.)were seeded in 12-well plates at a density of 140,000 cells per well.Increasing doses of mOX40L_miR122 (SEQ ID NO: 66) formulated in LNPswere added to each well directly after seeding the cells. Doses ofmOX40L_miR122 included 6.3 ng, 12.5 ng, 25 ng, or 50 ng mRNA per well.Control cells were either mock-treated or treated with negative controlmRNA (non-translatable version of the same mRNA containing multiple stopcodons).

Following treatment, cell surface expression of OX40L was detected usingflow cytometry. Cells were harvested by transferring the supernatants toa 96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells from each well were then lifted with trypsin-freechelating solution, and stained with PE-conjugated anti-mouse OX40Lantibody (R&D Systems, Minneapolis, Minn.) and visualized by flowcytometry. The results are shown in FIG. 2.

C. Results

FIG. 2 shows a dose-dependent expression of OX40L on the surface ofB16F10 cancer cells after treatment with OX40L modified mRNA. All fourdoses of mOX40L_miR122 generated significant OX40L expression on thecell surface compared to control samples.

These results show that administering an OX40L modified mRNA results inexpression of an OX40L polypeptide on the surface of target cells.

Example 2: In Vitro Expression Kinetics of OX40L on Cell Surface

In this example, expression levels of an OX40L polypeptide on thesurface of cancer cells were measured over time. Quantitation of OX40Lprotein expression was also measured.

A. Formulation of mOX40L_miR122

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L or human OX40L) and further comprising a miRNA bindingsite (miR-122) was used in this example (mOX40L_miR122, SEQ ID NO: 66;hOX40L_miR122, SEQ ID NO: 65). The OX40L modified mRNA was formulated ineither lipid nanoparticles (LNP) as described above in Example 1 orformulated in LIPOFECTAMINE 2000 (L2K) (ThermoFisher Scientific,Waltham, Mass.) according to the manufacturer's instructions.

B. Cell Lines

Human cervical carcinoma cells (HeLa, ATCC No. CCL-2; ATCC, Manassas,Va.) were seeded at a density of 250,000 cells per well in 6-wellplates. 24-hours post-seeding, L2K-formulated mOX40L_miR122 orhOX40L_miR122 containing 3 μg of mRNA was added to each well. The cellswere treated with mOX40L_miR122 or hOX40L_miR122 in the presence orabsence of 50 μg/ml mitomycin C 24 hours post-transfection.

Mouse colon adenocarcinoma cells (MC-38; Rosenberg et al., Science233(4770):1318-21 (1986)) were seeded at a density of 300,000 cells perwell in 6-well plates. LNP-formulated mOX40L_miR122 containing 3 μg ofmRNA was added to each well 24 hours after seeding the cells. The MC-38cells were treated with mOX40L_miR122 in the presence or absence of 25μg/ml mitomycin C.

Control cells were mock-treated. Cell surface expression of OX40L wasmeasured on Days 1, 2, 3, 5, and 7 following treatment withmOX40L_miR122 and Days 1, 2, 3, 4, and 5 following treatment withhOX40L_miR122. Cells were harvested and analyzed by flow cytometry asdescribed above in Example 1. The results for cells treated withmOX40L_miR122 are shown in FIG. 3A-3D; the results for cells treatedwith hOX40L_miR122 are shown in FIG. 3E. Cell lysates and cell culturesupernatants were also harvested and analyzed for OX40L proteinexpression (quantitated in nanograms per well). The results for mouseand human OX40L protein quantitation following treatments are shown inFIGS. 3F and 3G, respectively.

C. Results

FIG. 3A-3D shows that OX40L was detected on the surface of HeLa cellsout to at least Day 7 after treatment with mOX40L_miR122. FIG. 3A-3Dalso shows that cell surface expression of OX40L on MC-38 cells treatedwith mOX40L_miR122 returned to baseline by Day 5 after treatment. Inboth cell lines, the gradual reduction in cell surface expression levelsof OX40L over time was blocked by the presence of mitomycin C. FIG. 3Eshows that human OX40L expression was detected on the surface of HeLacells out to at least Day 5 after treatment with hOX40L_miR122.

No significant shedding of the OX40L polypeptide was detected in culturesupernatants. This suggests that the OX40L expressed from mRNA was notactively shed from the cell surface, which was confirmed in FIGS. 3F and3G. Twenty-four hours after treatment with mOX40L_miR122, hOX40L_miR122,or mock treatment, cell lysates were prepared using standard cell lysisbuffers and methods for protein analysis. FIG. 3F and FIG. 3G show thatboth mOX40L_miR122 (FIG. 3F) and hOX40L_miR122 (FIG. 3G) producedproteins that were recognized by commercially available ELISAs. Themajority of the expressed protein was associated with the cell lysate,with only approximately 0.1% of the produced protein detected in thesupernatant of transfected cells.

These results show that treatment of cells with an OX40L modified mRNAresults in expression of an OX40L polypeptide on the surface of targetcells. These results also show that only minor amounts of protein areshed from transfected cells.

Example 3. In Vitro Biological Activity of OX40L

T-cell activation involves two concurrent cell signaling events: aprimary signal from the T-cell receptor complex (e.g., CD3 stimulation)and a second signal from a costimulatory ligand-receptor interaction(e.g., OX40L/OX40R interaction). Kober et al., European Journal ofImmunology 38:2678-2688 (2008). In this example, the costimulatorybiological activity of OX40L expressed on the surface of cells treatedwith mOX40L_miR122 or hOX40L_miR122 was assessed.

A. Preparation of OX40L-Expressing Cells

Mouse melanoma cells (B16F10, ATCC No. CRL-6475; ATCC, Manassas, Va.)were seeded in 6-well plates at a density of 300,000 cells per well.Human cervical carcinoma cells (HeLa) were seeded in 6-well plates asdescribed above. A polynucleotide comprising an mRNA encoding an OX40Lpolypeptide and further comprising a miR-122 binding site (mouse OX40L,mOX40L_miR122, SEQ ID NO: 66; human OX40L, hOX40L_miR122, SEQ ID NO: 65)was formulated in L2K as described above in Example 2. 24 hours afterseeding the cells, formulations containing 3 μg of mOX40L_miR122 orhOX40L_miR122 mRNA were added to each well. Control cells were eithermock-treated or treated with negative control mRNA (non-translatableversion of the same mRNA except with no initiating codons). The cellswere incubated for 24 hours at 37° C.

B. Preparation of Naïve CD4+ T-Cells

Spleens from C57BL/6 mice were removed and processed using standardtechniques in the art to generate single cell suspensions ofsplenocytes. Total CD4⁺ T-cells were isolated from the splenocytesuspensions using a mouse CD4 T cell isolation kit (Miltenyi, San Diego,Calif.). Naïve human CD4⁺ T-cells were isolated from human peripheralblood mononuclear cells (PBMCs) by depleting non-CD4 cells using acommercially available magnetic bead T cell isolation kit.

C. T-Cell Activation Assay

200,000 T-cells were added to each well of transfected B16F10 cells orHeLa cells in the presence of agonistic anti-mouse CD3 antibody (R&DSystems, Minneapolis, Minn.) or agonistic anti-human CD3 antibody andsoluble anti-human CD28; and the cells were co-cultured for 72 hours(mouse) or 120 hours (human). A schematic of the assays is shown in FIG.4A.

After co-culture with T-cells, mouse IL-2 production was measured usinga mouse IL-2 ELISA. (mouse IL-2 DuoSet ELISA, R&D Systems, Minneapolis,Minn.). The amount of IL-2 produced by the CD4⁺ T-cells serves as anindicator of T-cell activation. Results are shown in FIG. 4B. Human IL-2production was measured using a human IL-2 ELISA (human IL-2 DuoSetELISA, R&D Systems, Minneapolis, Minn.). Results are shown in FIGS. 4C,4D, and 4E.

D. Results

FIG. 4B shows that OX40L expression on the surface of B16F10 cellstreated with mOX40L_miR122 elicits a T-cell IL-2 response in vitro. ThemOX40L_miR122 mRNA induced about 12 ng/ml of IL2. B16F10 cells treatedwith non-translated negative control mRNA showed baseline levels ofT-cell activation comparable to mock-treated cells (i.e., about 6 ng/mlof IL2). Therefore, the mOX40L_miR122 mRNA induced about two fold higherIL2 expression compared to a control (mock treated or non-translatedmRNA).

FIGS. 4C and 4D show that, in the presence of plate-coated anti-humanCD3 antibody and soluble anti-human CD28 as the primary T-cellactivators, co-culture with the OX40L mRNA transfected HeLa cellsgreatly enhanced IL-2 production. Without OX40L expression, little to noIL-2 production was detected. FIG. 4E shows a similar level of increasedhuman IL-2 production when the same experiment was performed withpre-stimulated (i.e., non-naïve) CD4⁺ T-cells.

These results show that the OX40L polypeptide is biologically active asa costimulatory molecule.

Example 4. In Vivo Expression Levels of Modified mRNA

To investigate in vivo expression levels of a polynucleotide comprisingmodified mRNA, a polynucleotide comprising an mRNA encoding luciferaseand further comprising a miR-122 binding site was prepared (SEQ ID NO:69). The luciferase modified mRNA was formulated in MC3 LNP. (USPublication no. US20100324120).

A. MC-38 Colon Adenocarcinoma Mouse Model

MC-38 colon adenocarcinoma tumors were established subcutaneously inC57BL/6 mice. (Rosenberg et al., Science 233(4770):1318-21 (1986)).

B. Treatment with Luciferase Modified mRNA

Once the MC-38 tumors reached approximately 200 mm³, mice were treatedwith a single intratumoral dose of 3.125 μg, 6.25 μg, 12.5 μg, 25 μg, or50 μg of luciferase modified mRNA (SEQ ID NO: 69; Cap1, G5 RP mRNA in1.5% DMG MC3 LNP). Control animals were treated with intratumoral doseof PBS. 24 hours post-treatment, animals were anesthetized, injectedwith the luciferase substrate D-luciferin and the bioluminescenceimaging (BLI) from living animals was evaluated in an IVIS imager 15minutes later. Signals from tumor tissue were obtained and compared withsignals from liver tissue in the same animal. Results are shown in FIG.5.

C. Results

FIG. 5 shows that the luciferase signal in tumor tissue was detected outto 48 hours post-dosing. FIG. 5 also shows that the three highest dosesof modified mRNA (50 μg, 25 μg, and 12.5 μg) yielded comparableluciferase signals in tumor tissue. The 12.5 μg dose of modified mRNAyielded a high tumor signal with a lower liver (normal tissue) signal inthe MC-38 colon carcinoma mouse model.

These results show that administration of a polynucleotide comprising amodified mRNA and a miRNA binding site preferentially targets tumortissues over normal tissues.

Example 5. In Vivo Dose-Dependent Expression of OX40L in B16F10 Tumors

In vivo expression of OX40L was assessed in a B16F10 tumor model.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared (mOX40L_miR122; SEQ ID NO: 66). The OX40L modified mRNA wasformulated in MC3 LNP as described in US20100324120. Negative controlmRNA was also prepared (OX40L_NST, SEQ ID NO: 68; a non-translatableversion of the same mRNA except no initiating codons).

B. Mouse Melanoma B16F10 Tumor Model

Subcutaneous B16F10 tumors were established in C57BL/6 mice. (Overwijket al. Current Protocols in Immunology Ch. 20, Unit 20.1 (2001)).

Once the tumor size reached approximately 200 mm³, animals were treatedwith a single intratumoral dose of mOX40L_miR122 (Cap1, G5 RP mRNA in0.5 mol % DMG MC3 LNP) at a dose of 5 μg mRNA (approximately 0.25 mg/kg)or 15 μg mRNA (approximately 0.75 mg/kg). Control animals were treatedwith equivalent doses of negative control mRNA, OX40L_NST. Additionalcontrol animals were treated with PBS.

C. Measurement of OX40L in Tumor Tissue

Animals were sacrificed 8 hours and 24 hours after dosing. Tumor tissuewas harvested and analyzed for expression of OX40L using a mouse OX40LELISA assay (R&D Systems, Minneapolis, Minn.). Results are shown in FIG.6 as the amount of OX40L present per gram of tumor tissue.

D. Results

FIG. 6 shows that a single intratumoral dose of 5 μg mOX40L_miR122resulted in over 200 ng OX40L/g tumor tissue at both 8 hours and 24hours post dosing. FIG. 6 also shows that a single intratumoral dose of15 μg mOX40L_miR122 resulted in over 500 ng OX40L/g tumor tissue at both8 hrs and 24 hours post-dosing.

In contrast, less than 100 ng OX40L was detectable in the liver ofanimals treated with the higher 15 μg dose of mOX40L_miR122.

These data show that administration of mOX40L_miR122 results insignificant levels of OX40L polypeptide expression in the tumor tissue.

Example 6. In Vivo Expression of OX40L in MC-38 Tumors

In vivo expression of OX40L was assessed in a MC-38 tumor model.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared (mOX40L_miR122; SEQ ID NO: 66). The OX40L modified mRNA wasformulated in MC3 LNP as described above. A negative control mRNA wasalso prepared (non-translatable version of the same mRNA containingmultiple stop codons; OX40L_NST; SEQ ID NO: 68).

B. MC-38 Colon Adenocarcinoma Mouse Model

MC-38 colon adenocarcinoma tumors were established subcutaneously inC57BL/6 mice. (Rosenberg et al., Science 233(4770):1318-21 (1986)).

Once the tumors reached a mean size of approximately 100 mm³, animalswere treated with a single intratumoral dose of mOX40L_miR122 (Cap1, G5RP mRNA in 1.5 mol % DMG MC3 LNP) at a dose of 12.5 μg mRNA. Controlanimals were treated with an equivalent dose of negative control mRNA,OX40L_NST. Additional control animals were left untreated (“NT”). Fordose-response experiments, animals were administered an intratumoralinjection of 3.125, 6.25, or 12.5 μg mOX40L_miR122; control animals wereleft untreated or treated with 12.5 μg negative control mRNA.

C. Measurement of OX40L in Tumor Tissue

To measure OX40L expression over time, animals were sacrificed 3, 6, 24,48, 72, and 168 hours after dosing. Tumor tissue was harvested andanalyzed for expression of OX40L using ELISA (R&D Systems, Minneapolis,Minn.), as described above in Example 5. Results are shown in FIG. 7A asthe amount of OX40L present per gram of tumor tissue.

To measure OX40L expression as a function of dose-response, animals weresacrificed 24 hours after dosing and tumor tissue was harvested foranalysis as described above. Tumor tissue, liver tissue, and spleentissue were analyzed for quantity of OX40L protein (FIG. 7B-7D, upper)and mRNA (FIG. 7B-7D, lower).

Tumor cells were also analyzed for expression of OX40L on the cellsurface using flow cytometry (data not shown). Tumor tissue was mincedand processed through cell strainers to prepare single cell suspensions.Cell suspensions were stained with PE-conjugated anti-mouse OX40Lantibody (R&D Systems, Minneapolis, Minn.), and visualized by flowcytometry.

D. Results

FIG. 7A shows that a single intratumoral dose of 12.5 μg mOX40L_miR122resulted in up to 1200 ng OX40L/g tumor tissue at 24 hours post dosing.The optical densities for two of the 24-hour OX40L-treated samples wereabove the standard range, resulting in underestimated values shown inFIG. 7A. FIG. 7A also shows OX40L expression was detectable in tumortissue out to 168 hours (7 days) post dosing. In contrast, controltreated animals showed no detectable OX40L in tumor tissue at any timepoint. FIG. 7B shows a dose-dependent increase in OX40L protein (upper)and mRNA (lower) in tumor tissue. FIGS. 7C and 7D show the presence ofOX40L protein and mRNA in liver and spleen (respectively) are lower thanthe amounts present in the tumor tissue.

Flow cytometry results showed that approximately 6.5% of all live,tumor-associated cells were positive for OX40L expression (data notshown).

These data show that administration of mOX40L_miR122 results insignificant levels of OX40L polypeptide expression in the tumor tissue.

Example 7. In Vivo Efficacy of OX40L Modified mRNA in a ColonAdenocarcinoma Model

In vivo efficacy of a polynucleotide comprising an mRNA encoding anOX40L polypeptide was assessed.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). A negativecontrol mRNA was also prepared (non-translatable version of the samemRNA containing multiple stop codons; NT OX40L_miR122; SEQ ID NO: 68).Both modified mRNAs were formulated in MC3 LNP as described above.

B. MC-38 Colon Adenocarcinoma Mouse Model

MC-38 colon adenocarcinoma tumors were established subcutaneously inC57BL/6 mice as described above.

Fourteen days after tumor cell inoculation, animals were treated twiceweekly for three weeks with an intratumoral dose of MC3 LNP-formulatedmodified mRNA (15 μg mRNA per dose). Control animals were treated withan equivalent dose and regimen of negative control mRNA, NT OX40L_miR122(SEQ ID NO: 68).

Tumor volume was measured at the indicated time points using manualcalipers. Tumor volume was recorded in cubic millimeters.

The in vivo efficacy study was carried out through Day 42 post-dosing.At the completion of the study, the full data sets were analyzed andpresented in FIGS. 8A and 8B. Final Kaplan-Meier survival curves wereprepared and are shown in FIG. 8C. Endpoints in the study were eitherdeath of the animal or a tumor volume reaching 1500 mm³.

C. Results

FIG. 8A shows that administering a control modified mRNA had littleeffect on the tumor volume, as assessed at the study completion (Day 42after the first dose). FIG. 8B shows that administering mOX40L_miR122 tothe mice inhibited or slowed tumor growth in some animals and reduced ordecreased the size of the tumor in some animals, as assessed at studycompletion (Day 42).

FIG. 8C shows that animals receiving mOX40L_miR122 had longer survivaltimes as measured on Day 42 compared to control animals.

These data show that mOX40L_miR122 polynucleotides have anti-tumorefficacy when administered in vivo.

Example 8. In Vivo Expression of OX40L in A20 Tumors

Mouse models of B-cell lymphoma using the A20 cell line are useful foranalyzing a tumor microenvironment. (Kim et al., Journal of Immunology122(2):549-554 (1979); Donnou et al., Advances in Hematology 2012:701704(2012)). Therefore, in vivo expression of OX40L and the tumormicroenvironment were assessed in an A20 B-cell lymphoma tumor model.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). The OX40Lmodified mRNA was formulated in MC3 LNP as described above. A negativecontrol mRNA was also prepared (non-translatable version of the samemRNA containing multiple stop codons; NT OX40L; SEQ ID NO: 68).

B. A20 B-Cell Lymphoma Tumor Model

B-cell lymphoma tumors were established subcutaneously in BALB/c mice.Mouse B-cell lymphoma cells (A20, ATCC No. TIB-208; ATCC, Manassas, Va.)were cultured according to the vendor's instructions. Cells wereinoculated subcutaneously in BALB/c mice to generate subcutaneoustumors. Tumor was monitored for size and palpability.

Once the tumors reached a mean size of approximately 1300 mm³, animalswere separated into two groups. Group I was treated with a singleintratumoral dose of mOX40L_miR122 (Cap1, G5 RP mRNA in 0.5 mol % DMGMC3 LNP) at a dose of 15 μg mRNA. Group II (controls) was treated withan equivalent dose of negative control mRNA, NT OX40L.

C. Measurement of OX40L in Tumor Tissue

Tumor tissue was harvested 24 hours after dosing and analyzed forexpression of OX40L using ELISA (R&D Systems, Minneapolis, Minn.), asdescribed above in Example 5. Results are shown in FIG. 9A as the amountof OX40L present per gram of tumor tissue.

A20 tumor cells were also analyzed for cell surface expression of OX40L.Tumor tissue was minced and processed through cell strainers to preparesingle cell suspensions. Cells were stained with anti-mouse OX40Lantibody (goat IgG polyclonal, PE conjugated; R&D Systems, Minneapolis,Minn.) and anti-mouse CD45 antibody (clone 30-F11, PE-Cy5 conjugated;eBioscience, San Diego, Calif.) to identify leukocytes (i.e., A20 cancercells and infiltrating immune cells). The cells were subsequentlyanalyzed by flow cytometry. Results are shown in FIGS. 9B and 9C.

D. Results

FIG. 9A shows that a single intratumoral dose of 15 μg mOX40L_miR122resulted in up to 250 ng OX40L/g tumor tissue at 24 hours after dosing.In contrast, control treated animals showed less than 100 ng OX40L intumor tissue 24 hours after dosing.

FIG. 9B shows that approximately 3% of all live, CD45⁺ cells (i.e.,tumor cells) expressed OX40L on the cell surface. In a similarexperiment, approximately 15.8% total live cells from the tumor werefound to express introduced OX40L, compared to less than 0.5%OX40L-positive live cells in tumors treated with the negative controlmRNA (FIG. 9C).

These data show that administration of mOX40L_miR122 results insignificant levels of OX40L polypeptide expression in the tumor tissue.

Example 9. In Vivo Pharmacodynamic Effects of OX40L

The ability of mOX40L_miR122 mediated OX40L expression to recruitnatural killer (NK) cells to the tumor site was assessed.

A. A20 B-Cell Lymphoma Tumor Model

The B-cell lymphoma tumors described above in Example 8 were alsoassessed for NK cell infiltration following treatment. As describedabove, mice were treated with a single intratumoral dose of eithermOX40L_miR122 or control NT OX40L mRNA (15 μg dose; Cap1, G5 RP mRNA in0.5 mol % DMG MC3 LNP). 24 hours after dosing, tumors were harvested asdescribed above and processed through cell strainers to prepare singlecell suspensions.

B. Natural Killer Cell Infiltration

Single cell suspensions were incubated with anti-mouse NKp46 antibody(clone 29A1.4, PerCP-eFluor® 710 conjugated; eBioscience, San Diego,Calif.), which is specific to the NK cell marker p46 (CD335), andanti-mouse CD3 antibody (clone 145-2C11, FITC conjugated; BioLegend, SanDiego, Calif.), which is specific to T-cells. The cells were analyzedbased on CD45⁺ expression for leukocyte, as well as NKp46 and CD3εexpression using flow cytometry. NK cells are p46⁺ and CD3. Results areshown in FIGS. 10A and 10B.

C. Results

FIG. 10A shows that animals treated with mOX40L_miR122 exhibitedapproximately 5-fold increase in the relative number of NK cells withinA20 tumors 24 hours after dosing. FIG. 10B shows the individual animaldata from the same study.

These results show that treatment with a polynucleotide comprising anmRNA encoding an OX40L polypeptide increased the number of NK cellswithin the tumor microenvironment.

Example 10. In Vivo Efficacy of OX40L Modified mRNA in a B-Cell LymphomaModel

In vivo efficacy of mOX40L_miR122 was assessed in a B-cell lymphomamodel.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). A negativecontrol mRNA was also prepared (non-translatable version of the FactorIX mRNA containing multiple stop codons; NST-FIX, SEQ ID NO: 67). Bothmodified mRNAs were formulated in the same manner (Cap1, G5 RP mRNA in1.5 mol % DMG MC3 LNP).

B. A20 B-Cell Lymphoma Tumor Model

B-cell lymphoma tumors were established subcutaneously in BALB/c mice.Mouse B-cell lymphoma cells (A20, ATCC No. TIB-208; ATCC, Manassas, Va.)were cultured according to the vendor's instructions. Cells wereinoculated subcutaneously in BALB/c mice to generate subcutaneoustumors. Tumor was monitored for size and palpability.

Once the tumors reached a mean size of approximately 100 mm³, animalswere separated into two groups. Group I was treated with repeatedintratumoral doses of mOX40L_miR122 (Cap1, G5 RP mRNA in 1.5 mol % DMGMC3 LNP) at a dose of 12.5 μg mRNA. Group II (control) was treated withan equivalent dose of negative control mRNA, NST-FIX. Animals were dosedon Days 20, 23, 27, 30, 34, 37, 41, 44, 48, and 51. Results are shown inFIGS. 11A, 11B, 11C, and 11D.

The study was carried out through Day 57. Final Kaplan-Meier survivalcurves were prepared and are shown in FIG. 11D. Endpoints in the studywere either death of the animal or a tumor volume reaching 2000 mm³.

C. Results

FIG. 11A shows individual tumor growth in animals treated with controlNST-FIX mRNA. FIG. 11B shows individual tumor growth in animals treatedwith mOX40L_miR122. Arrows represent dosing days. Multiple doses of acontrol modified mRNA had little effect on the tumor volume. Incontrast, multiple doses of mOX40L_miR122 reduced or decreased the sizeof tumors in some animals or inhibited the growth of tumors in someanimals.

FIG. 11C shows the average tumor size for each group as assessed at Day35 of the study. These data show that administering mOX40L_miR122reduced or inhibited tumor growth compared to treatment with controlmRNA. The following formula was used to calculate the percentage oftumor growth inhibition (TGI) at Day 34 compared to Day 19:

TGI %=[(Vc−Vt)/Vc−Vo)]×100

Using the formula above and the data shown in FIG. 11C, the TGI % formOX40L_miR122 was 57%. In other words, animals treated withmOX40L_miR122 showed 57% tumor growth inhibition between Days 19 and 34compared to control treated animals.

FIG. 11D shows that animals receiving mOX40L_miR122 had longer survivaltimes as measured on Day 42 compared to control animals.

These data show that mOX40L_miR122 polynucleotides have anti-tumorefficacy when administered in vivo.

Example 11. In Vivo Memory Immune Response

mOX40L_miR122 was assessed for its ability to induce an adaptive(memory) immune response in the MC-38 adenocarcinoma model.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). A negativecontrol mRNA was also prepared (non-translatable version of the OX40LmRNA containing multiple stop codons; NST-OX40L, SEQ ID NO: 68). Bothmodified mRNAs were formulated in the same manner (Cap1, G5 RP mRNA in1.5 mol % DMG MC3 LNP).

B. MC-38 Colon Adenocarcinoma Model

MC-38 colon adenocarcinoma tumors were established subcutaneously inC57BL/6 mice as described above.

Seven days after tumor cell inoculation, animals were treated everythree days (Q3D) for a maximum of 10 intratumoral doses of MC3LNP-formulated modified mRNA (12.5 μg mRNA per dose). Control animalswere treated with an equivalent dose and regimen of negative controlmRNA, NT OX40L_miR122.

Tumor volume was measured at the indicated time points using manualcalipers. Tumor volume was recorded in cubic millimeters. At Day 60post-tumor inoculation, six apparent complete responder animals (CR)from the mOX40L_miR122 group were re-challenged with 5×10⁵ MC-38 tumorcells; as a control, six naïve animals were also inoculated with 5×10⁵MC-38 cells. The results of the analysis are shown in FIGS. 12A and 13B.

C. Results

FIG. 12A shows individual tumor growth in animals treated with controlNST-OX40L mRNA, mOX40L_miR122, or PBS. FIG. 12A shows that 6 out of 15animals administered mOX40L_miR122 (40%) exhibited a complete responsewith no significant tumor growth as measured on Day 60. In comparison,animals administered the negative control mRNA construct or PBS showedsignificant tumor growth through Day 60. (FIG. 12A). These results showthat administering an mRNA encoding an OX40L polypeptide reduces ordecreases the size of a tumor or inhibits the growth of a tumor.

At Day 60, six complete responders (“CR”) from the mOX40L_miR122 groupand six naïve control animals were re-challenged with MC-38 cells. FIG.12B shows individual tumor growth in animals re-challenged with MC-38cells. Animals previously administered mOX40L_miR122 showed no tumorgrowth (0/6 animals) for 23 days after re-challenge with tumor cells. Incomparison, 67% (6/9 animals) of the animals in the naïve control groupshowed tumor growth at Day 23. These results show that administering anmRNA encoding an OX40L polypeptide induces a memory immune response withanti-tumor effects.

Example 12. Sustained In Vivo Expression of OX40L in A20 Tumors

In vivo expression of OX40L in the tumor microenvironment was assessedin an A20 B-cell lymphoma tumor model at various timepoints after oneand/or two doses of a polynucleotide comprising an mRNA encoding anOX40L polypeptide.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). The OX40Lmodified mRNA was formulated in MC3 LNP as described above. A negativecontrol mRNA was also prepared (non-translatable version of the samemRNA containing multiple stop codons; NST-OX40L; SEQ ID NO: 68).

B. A20 B-Cell Lymphoma Tumor Model

B-cell lymphoma tumors were established as described above. Once thetumors reached a mean size of approximately 1300 mm³, animals wereseparated into three groups. Group I was treated with a singleintratumoral dose of mOX40L_miR122 (Cap1, G5 RP mRNA in 0.5 mol % DMGMC3 LNP) at a dose of 15 μg mRNA. Group II (control) was treated with anequivalent dose of negative control mRNA, NT OX40L. Group III wastreated with an intratumoral injection of PBS. Each group also compriseda sub-group of animals that received a second dose of mRNA or PBS 7 daysafter the first dose.

C. Measurement of OX40L Expression

Live cells from A20 tumor cells were analyzed for cell surfaceexpression of OX40L. Tumor tissue was minced and processed through cellstrainers to prepare single cell suspensions. Live cells were stainedwith anti-mouse OX40L antibody (goat IgG polyclonal, PE conjugated; R&DSystems, Minneapolis, Minn.). The cells were subsequently analyzed byflow cytometry. Results are shown in FIG. 13.

D. Results

FIG. 13 shows statistically significant OX40L expression at 24 hours, 72hours, and 7 days after a single dose of mOX40L_miR122. In particular,FIG. 13 shows that OX40L expression in A20 tumors is sustained at 72hours and 7 days after a single dose of mOX40L_miR22. In animalsreceiving a second dose, statistically significant OX40L expression wasdetected 24 hours after the second dose of mOX40L_miR122.

These data show that administration of mOX40L_miR122 results insignificant, sustained levels of OX40L polypeptide expression in thetumor tissue.

Example 13. Identity of Cell Types Expressing OX40L after mRNA Treatment

The identity of cell types expressing OX40L post-mRNA treatment withinA20 and MC38 tumors was evaluated. A polynucleotide comprising an mRNAencoding an OX40L polypeptide (murine OX40L) and further comprising amiRNA binding site (miR-122) was prepared as described above. Mousemodels of A20 tumors and MC38 tumors were established as describedabove.

A. Cell Differentiation by Flow Cytometry

Cells within A20 tumors were differentiated by CD19 and CD45 antibodies,which identify CD19-expressing B-lymphoma A20 cancer cells (CD19⁺,CD45⁺) from the non-cancer immune infiltrates (CD19⁻, CD45⁺) and thenon-cancer/nonimmune cells (CD19⁻, CD45⁻), respectively. Results areshown in FIG. 14A. Cells within MC38 tumors were differentiated by CD45marker to differentiate infiltrating host immune cells (CD45⁺) fromcancer cells and non-immune host cells (CD45⁻). Results are shown inFIG. 14B. Immune infiltrate cells were differentiated with CD11bantibody. CD11b⁺ immune infiltrate cells were separately analyzed forOX40L expression. Results are shown in FIG. 14C.

B. Results

FIG. 14A shows that in A20 tumors treated with mOX40L_miR122, 76% of theOX40L expressing cell population were the A20 tumor cells themselves,whereas approximately 19% of the OX40L positive cell population wereinfiltration immune cells within the A20 tumors. The population of OX40Lexpressing host immune cells was shown to be predominantly myeloidlineage cells, as determined by positive staining for CD11b. Of theCD11b⁺ myeloid lineage cells in the A20 tumors, an average of 25.4% werepositive for OX40L expression (FIG. 14C).

FIG. 14B shows that in MC38 tumors, the majority of OX40L positive cellswere cancer cells (an average of 57.3%), while 35.6% of the positivecells were immune infiltrates, again primarily derived from myeloidlineage (CD45⁺, CD11b⁺).

These data show that administration of mOX40L_miR122 results in OX40Lexpression in a significant percentage of the tumor environmentpost-intratumoral mRNA administration, and that a majority ofOX40L-expressing cells were cancer cells followed by myeloid immune cellinfiltrates.

Example 14. Modulation of Immune Cell Populations within Tumors Treatedwith OX40L mRNA

Given the demonstrated activity of OX40L on innate immune natural killer(NK) cells and adaptive CD4+/CD8+ T cells, the objective of thefollowing studies was to evaluate the pharmacodynamic effects of OX40Lintratumoral treatment on tumor-associated immune cell populations.Mouse A20 and MC38 tumor models were established as described above.

A. Cell Differentiation by Flow Cytometry

A20 tumors were treated with a single 12.5 μg dose of mOX40L_miR122 orcontrol mRNA (RNA/LNP) formulated in lipid nanoparticles. Tumor sampleswere initially analyzed 24 hours following treatment. NK cells weredifferentiated using an antibody against the mature NK cell surfacemarker, DX5. Results are shown in FIG. 15A. Other tumor samples wereanalyzed 14 days after treatment with mOX40L_miR122. CD4⁺ and CD8⁺T-cells were identified using anti-mouse CD4 and anti-mouse CD8antibodies, respectively. Results are shown in FIG. 15B-15C.

A similar experiment was performed in the MC38 tumor model. Mice withMC38 tumors were administered a single intratumoral injection ofmOX40L_miR122 or NST-OX40L. In some animals a second dose of mRNA wasadministered 6 days after the first dose. Immune cell infiltrate wasassessed for CD8⁺ cells 24 hours and 72 hours after each dose of mRNA.Results are shown in FIG. 15D.

B. Results

FIG. 15A shows that 24 hours after administration of mOX40L_miR122 toA20 tumors, NK cells infiltration significantly increased in OX40L-dosedanimals compared to controls. FIG. 15B-15C show that 14 days afteradministration of mOX40L_miR122 to A20 tumors, both CD4⁺ (FIG. 15B) andCD8⁺ (FIG. 15C) T-cell infiltration into the tumor microenvironmentsignificantly increased compared to control tumor samples.

FIG. 15D shows a significant increase in infiltrating CD8⁺ T-cells 72hours after a second dose of mOX40L_miR122 in MC38 tumors compared tocontrol treated tumors.

These data from two tumor models demonstrate that administration of apolynucleotide comprising an mRNA encoding an OX40L polypeptide promotesincreased numbers of both innate and adaptive immune cells within thetumor microenvironment.

Example 15. In Vivo Efficacy in A20 Tumors

In vivo efficacy was assessed in the A20 tumor model. A20 tumors wereestablished as described above.

A. Tumor Treatment

Mice were treated with either 12.5 μg per dose mOX40L_miR122 in LNP,12.5 μg per dose negative control mRNA designed not to be translatedinto protein in LNP (NST-OX40L), a PBS negative control, or leftuntreated. mRNA/LNPs and negative controls were dosed in a 25 μl volumedirectly into the A20 tumor lesions at a frequency of once every 7 daysfor up to 6 maximum doses. The tumor volumes of individual animals areshown in FIG. 16A (measured as mm³). A Kaplan-Meier survival curve ofthe same animals is shown in FIG. 16B. The x-axes of both graphs areDays post disease induction, i.e. subcutaneous cancer cell implantation.

B. Results

FIG. 16A shows that an increased number of animals treated withmOX40L_miR122 exhibited tumor growth inhibition compared to controlanimals. All of the control animals (30/30) were sacrificed by Day 60post disease induction (primarily due to reaching the pre-determinedtumor burden endpoint ≥2000 mm3). In contrast, 4/9 animals or 44% of themOX40L_miR122-treated mice had not yet reached the tumor burden endpointby Day 98.

FIG. 16B shows the survival benefit of mOX40L_miR122 treatment, in which2 mice in the mOX40L_miR122 arm (as designated by asterisk * in FIG.16A) and 1 from the PBS group were removed from the study due to tumorulceration and not included in the survival estimate. By this criteria,2/8 or 25% of the OX40L mRNA treated animals were apparent completeresponders by Day 98 post implantation compared to 0/29 of the controlanimals.

These data show the in vivo efficacy of administering a polynucleotidecomprising an mRNA encoding an OX40L polypeptide (mOX40L_miR122) in theA20 tumor model.

Example 16. miR-122 Modulates OX40L Expression

The effects of incorporating a miR-122 binding site into thepolynucleotide were assessed.

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mouse OX40L, mOX40L_miR122, SEQ ID NO: 66;human OX40L, hOX40L_miR122, SEQ ID NO: 65). Polynucleotides comprisingan mRNA encoding mouse OX40L polypeptide or human OX40L polypeptide,each without a miR-122 binding site, were also prepared to compare theeffects of the presence of the miR-122 binding site.

B. Cell Transfections

Primary human hepatocytes, human liver cancer cells (Hep3B), and humancervical carcinoma cells (HeLa) were transfected with a polynucleotidecomprising an mRNA encoding human OX40L polypeptide (hOX40L) or apolynucleotide comprising an mRNA encoding human OX40L polypeptide andfurther comprising a miR-122 binding site (hOX40L_miR122). Cells wereanalyzed for OX40L expression 6 hours, 24 hours, and 48 hours aftertransfection. Results are shown in FIG. 17A. The same experiment wasalso performed with mouse OX40L, as shown in FIG. 17B.

C. Results

FIG. 17A shows that incorporating a miR-122 binding site into thepolynucleotide markedly reduced human OX40L expression in primaryhepatocytes at later timepoints. Specifically, at 24 hourspost-transfection, OX40L expression was reduced by 88% from 6,706ng/well in cells treated with a hOX40L (no miR-122 binding site) to 814ng/well in cells treated with hOX40L_miR122 (comprising a miR-122binding site). At 48 hours post-transfection, OX40L expression wasreduced by 94% from 11,115 ng/well to 698 ng/well in cells treated witha polynucleotide comprising a miR-122 binding site.

FIG. 17B shows similar results for mouse OX40L. Incorporating a miR-122binding site into the polynucleotide reduced mouse OX40L expression inprimary hepatocytes by 85% at 24 hours (from 1,237 ng/well to 182ng/well) and by 91% at 48 hours (from 1,704 ng/well to 161 ng/well).

These data show that incorporating a microRNA binding site (miR-122)into a polynucleotide comprising an mRNA encoding an OX40L polypeptidereduces expression of the OX40L polypeptide in primary hepatocytescompared to a polynucleotide lacking a miR-122 binding site.

Example 17. In Vivo Activity of an OX40L-Encoding PolynucleotideFollowing Intravenous Administration

A. Preparation of OX40L Modified mRNA

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). A negativecontrol mRNA was also prepared (non-translatable version of the samemRNA containing multiple stop codons: NST_OX40L_122).

B. Acute Myeloid Leukemia (AML) Tumor Model

Acute myeloid leukemia (AML) tumors were established subcutaneously inmice. Mouse AML cells (P388D1 cells, ATCC No. CCL-46, ATCC, Manassas,Va.) were cultured according to the vendor's instructions. Cells wereinoculated subcutaneously in DBA/2 mice to generate subcutaneous tumors.Tumors were monitored for size and palpability.

Once the tumors were established, animals were separated into fivegroups. Dosing for the intratumoral dosing groups was every 7 days(Q7D), beginning 7 days after tumor implantation. Group I was treatedwith intratumoral doses of mOX40L_miR122 at a dose of 12.5 μg mRNA(fixed dose). Group II was treated with intratumoral doses of controlNST_OX40L_122 mRNA at the same dosing regimen. Dosing for theintravenous dosing groups was every 7 days, beginning 4 days after tumorimplantation. Group III was treated with intravenous doses ofmOX40L_miR122 at a dose of 0.5 mg mRNA per kg body weight. Group IV wastreated with intravenous doses of control mRNA at the same dosingregimen. Group V was treated with intravenous doses of PBS.

C. Results

Results are shown in FIG. 18A-18E and FIG. 19. FIG. 18A shows individualtumor growth in animals treated with intratumoral doses of controlNST_OX40L_122 mRNA. FIG. 18B shows individual tumor growth in animalstreated with intratumoral doses of mOX40L_miR122 mRNA. FIG. 18C showsindividual tumor growth in animals treated with intravenous controlmRNA. FIG. 18D shows individual tumor growth in animals treated withintravenous doses of mOX40L_miR122 mRNA. FIG. 18E shows individual tumorgrowth in animals treated with intravenous doses of PBS (negativecontrol).

These results show that both intratumoral and intravenous administrationof a polynucleotide encoding an OX40L polypeptide comprising a miRNAbinding site reduces or inhibits tumor growth compared to control mRNAor PBS treatment.

FIG. 19 shows the survival curves for animals treated with intravenousdoses of mOX40L_miR122 compared to animals treated intravenously withcontrol mRNA or PBS. These results show that intravenous dosing ofmOX40L_miR122 increases survival in a mouse tumor model compared tosurvival of mice treated with a control mRNA.

Example 18. In Vivo Efficacy of Combination of an mRNA Encoding an OX40LPolypeptide, and an Anti-PD-1 Antibody

A. Preparation of OX40L Modified mRNA and Anti-PD-1 Antibody

A polynucleotide comprising an mRNA encoding an OX40L polypeptide(murine OX40L) and further comprising a miRNA binding site (miR-122) wasprepared as described above (mOX40L_miR122; SEQ ID NO: 66). A negativecontrol mRNA was also prepared (NST_OX40L_122).

Anti-PD-1 monoclonal antibody (BioXcell BE0146, anti-mPD-1, cloneRMP1-14, Lot No. 5792-599016J1) dosing solutions were prepared bydiluting an aliquot of the stock (6.37 mg/mL) to 0.5 mg/mL in sterilePBS. The 0.5 mg/mL dosing solution provided the 5 mg/kg dosage in adosing volume of 10 mL/kg. The anti-PD-1 dosing solution was preparedfresh daily and stored protected from light at 4° C.

Rat IgG2a (BioXcell BE0089, Rat IgG2a, clone 2A3, Lot No. 601416M1)dosing solutions were prepared by diluting an aliquot of the stock (7.38mg/mL) to 0.5 mg/mL in sterile PBS. The 0.5 mg/mL dosing solutionprovided the 5 mg/kg dosage in a dosing volume of 10 mL/kg. Theanti-PD-1 dosing solution was prepared fresh daily and stored protectedfrom light at 4° C.

B. MC38 Colon Adenocarcinoma Model

MC-38 colon adenocarcinoma tumors were established subcutaneously inC57BL/6 mice as described above.

Once the tumors were established, animals were divided into groups andreceived intratumoral doses of one of the following combinationtherapies shown in the table below:

TABLE 10 Combination Dosing and Interval Group Treatment Dose Interval iNST_OX40L_122 2.5 μg mRNA per dose Q7D Rat IgG2a antibody 5 mg per kgBIWx2 ii mOX40L_miR122 2.5 μg mRNA per dose Q7D Rat IgG2a antibody 5 mgper kg BIWx2 iii NST_OX40L_122 2.5 μg mRNA per dose Q7D Anti-PD-1antibody 5 mg per kg BIWx2 iv mOX40L_miR122 2.5 μg mRNA per dose Q7DAnti-PD-1 antibody 5 mg per kg BIWx2 v PBS NA Q7D Anti-PD-1 antibody 5mg per kg BIWx2 vi PBS NA Q7D Rat IgG2a antibody 5 mg per kg BIWx2

Mice received intratumoral doses of mRNA every 7 days (Q7D). Micereceived intratumoral doses of antibody every two weeks (BIWx2).

C. Results

Results are shown in FIG. 20A-20E and FIG. 21. FIG. 20A shows individualtumor growth in animals treated with intratumoral doses of controlNST_OX40L_122 mRNA combined with intratumoral doses of control antibody.There were 0/15 complete responders (CR) in the control group. FIG. 20Bshows individual tumor growth in animals treated with intratumoral dosesof mOX40L_miR122 mRNA combined with intratumoral doses of controlantibody. By Day 90 post-implantation, the CR was 0/15 for this group.FIG. 20C shows individual tumor growth in animals treated withintratumoral control mRNA combined with intratumoral doses of anti-PD-1antibody. By Day 90 post-implantation, the CR was 2/15 for this group.FIG. 20D shows individual tumor growth in animals treated withintratumoral doses of mOX40L_miR122 mRNA combined with intratumoraldoses of anti-PD-1 antibody. By Day 90 post-implantation, the CR was6/15 for the dual combination group. FIG. 20E shows individual tumorgrowth in animals treated with intratumoral doses of PBS combined withintratumoral doses of anti-PD-1 antibody. By Day 90 post-implantation,the CR was 0/15 for this group. FIG. 20F shows individual tumor growthin animals treated with intratumoral doses of PBS combined withintratumoral doses of control antibody. By Day 90 post-implantation, theCR for this treatment group was 0/14.

These results show that combination therapy comprising a polynucleotidecomprising an mRNA encoding an OX40L polypeptide and an immunecheckpoint inhibitor, such as an anti-PD-1 antibody, is effective invivo for inhibiting or reducing tumor growth in the MC38 mouse tumormodel. The combination of mOX40L_miR122 with anti-PD-1 antibody showedsynergistic in vivo anti-tumor efficacy. These results also show thatlower doses of mRNA can be used in combination therapy.

FIG. 21 shows the survival curves for animals in the same treatmentgroups. These results show that combining intratumoral dosing of amodified OX40L mRNA with intratumoral dosing of an anti-PD-1 antibodyeffectively increases survival in a mouse tumor model compared tocontrol treatment groups.

Example 19. In Vivo Memory Immune Response Following Treatment withCombination Therapy

Mice were treated with mOX40L_miR122 combined with anti-PD-1 antibody asdescribed above in Example 18. At Day 90 post-tumor inoculation, fourcomplete responder animals (CR) from the mOX40L_miR122+anti-PD-1combination therapy group were re-challenged with 5×10⁵ MC38 tumorcells. As a control, 10 naïve animals were also inoculated with 5×10⁵MC38 cells. The results of the analysis are shown in FIGS. 22A and 22B.

FIG. 22A shows individual tumor growth in naïve animals challenged withMC38 cells. Naïve mice began developing detectable tumors approximately5 days after implantation, and tumors continued to grow during thestudy. FIG. 22B shows individual tumor growth in the complete responderanimals previously given intratumoral doses of combination therapy ofmOX40L_miR122 and anti-PD-1 antibody. The complete responder animalsshowed no tumor growth (0/4 animals) for 23 days after re-challenge withtumor cells. In contrast, naïve animals showed a high percentage oftumor growth. These results show that intratumoral dosing of an mRNAencoding an OX40L polypeptide combined with an anti-PD-1 antibodyinduces a memory immune response with anti-tumor effects.

OTHER EMBODIMENTS

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes can be made withinthe purview of the appended claims without departing from the true scopeand spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the disclosure.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1.-17. (canceled)
 18. A method for treating cancer in a subject byinducing or enhancing an anti-tumor immune response, comprisingadministering to the subject a messenger RNA (mRNA) encoding an OX40Lpolypeptide, wherein the mRNA comprises a 3′ untranslated region (UTR)comprising at least one microRNA-122 (miR-122) binding site, therebytreating cancer in the subject by inducing or enhancing an anti-tumorimmune response.
 19. The method of claim 18, wherein the miR-122 bindingsite is a miR-122-3p binding site.
 20. The method of claim 18, whereinthe miR-122 binding site is a miR-122-5p binding site.
 21. The method ofclaim 20, wherein the miR-122-5p binding site comprises the nucleotidesequence as set forth in SEQ ID NO:
 26. 22. The method of claim 18,wherein the OX40L polypeptide comprises the amino acid sequence as setforth in SEQ ID NO:
 1. 23. The method of claim 18, wherein the mRNAcomprises an open reading frame, and wherein the open reading framecomprises a nucleotide sequence as set forth in SEQ ID NO: 4 or anucleotide sequence at least 90% identical to SEQ ID NO:
 4. 24. Themethod of claim 18, wherein the mRNA comprises a nucleotide sequence asset forth in SEQ ID NO: 65 or a nucleotide sequence at least 90%identical to SEQ ID NO:
 65. 25. The method of claim 18, wherein the mRNAis chemically modified.
 26. The method of claim 25, wherein the mRNA isfully modified with chemically-modified uridines.
 27. The method ofclaim 26, wherein the chemically-modified uridines areN1-methylpseudouridines (m1ψ).
 28. The method of claim 25, wherein themRNA is fully modified with 5-methylcytosine or is fully modified withN1-methylpseudouridines (m1ψ) and 5-methylcytosine.
 29. The method ofclaim 18, wherein the mRNA is formulated in a lipid nanoparticle. 30.The method of claim 29, wherein the lipid nanoparticle is administeredintratumorally.
 31. The method of claim 18, comprising administering aneffective amount of a PD-1 antagonist, a PD-L1 antagonist or a CTLA-4antagonist.
 32. The method of claim 31, wherein the PD-1 antagonist isan antibody or antigen binding portion thereof that specifically bindsto PD-1, wherein the PD-L1 antagonist is an antibody or antigen bindingportion thereof that specifically binds to PD-L1, and wherein the CTLA-4antagonist is an antibody or antigen binding portion thereof thatspecifically binds to CTLA-4.
 33. The method of claim 32, wherein thePD-1 antagonist is selected from the group consisting of nivolumab,pembrolizumab, and pidilizumab, wherein the PD-L1 antagonist is selectedfrom the group consisting of durvalumab, avelumab, and atezolizumab, andwherein the CTLA-4 antagonist is selected from the group consisting ofipilimumab and tremelimumab.
 34. The method of claim 18, wherein theanti-tumor immune response in the subject comprises T cell activationand wherein the T cell activation reduces or decreases the size of atumor, or inhibits growth of a tumor, in the subject.
 35. The method ofclaim 18, wherein the anti-tumor immune response in the subjectcomprises increasing the number of NK cells in the tumormicroenvironment.
 36. A lipid nanoparticle comprising a messenger RNA(mRNA) encoding an OX40L polypeptide, wherein the lipid nanoparticlecomprises about 20-60 mol % ionizable cationic lipid, about 5-25 mol %non-cationic lipid, about 25-55 mol % sterol, and about 0.5-15 mol %PEG-modified lipid.
 37. A composition comprising a messenger RNA (mRNA)encoding an OX40L polypeptide, and a pharmaceutically acceptablecarrier, wherein the mRNA comprises a 3′ untranslated region (UTR)comprising at least one microRNA-122 (miR-122) binding site.